Human signal transduction serine/threonine kinase

ABSTRACT

An isolated and purified human Ste20-like serine/threonine signal transduction kinase is described. A cDNA sequence which encodes the native signal transduction molecule is disclosed as well as the structural coding region and the amino acid residue sequence. Methods are provided which employ the sequences to identify compounds that modulate the biological and/or pharmacological activity of the transduction molecule and hence regulate cell physiology. Biologically-effective antisense molecules, as well as dominant negative mutant versions of the biomolecule are described which are suitable for therapeutic use. The invention is also drawn toward the diagnosis, prevention, and treatment of pathophysiological disorders mediated by the signal transduction molecule.

This is a Continuation in Part Application filed under 37 CFR 53(b) ofU.S. application Ser. No. 09/211,930, filed Dec. 15, 1998, which claimedpriority under 35 USC §119(a) from UK Application GB 9726851.0 entitledHUMAN SIGNAL TRANSDUCTION SERINE/THREONINE KINASE, filed Dec. 19, 1997;the entire disclosures of which are each incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to nucleic acid and amino acid sequencespertaining to a novel human signal transduction serine threonine proteinkinase. Molecular sequences are provided for the design and synthesis ofentities that modulate biological and/or pharmacological activity of thenative biomolecule. The sequences are also provided for employment toidentify compounds that modulate biological and/or pharmacologicalactivity of the native biomolecule. Biologically-effective antisensemolecules are provided, as well as dominant negative mutant versions ofthe signal transduction kinase which are suitable for therapeutic use.The invention is also drawn toward the study, prevention, diagnosis, andtreatment of pathophysiological disorders mediated by the novelbiomolecule.

BACKGROUND OF THE INVENTION

Cellular response mechanisms to stress are fundamentally important tothe human immune system. Stress responses represent carefully devisedcellular defense mechanisms which were developed at an early pointduring evolution; evidenced by the fact that biomolecules implicated instress response exhibit remarkable similarity across the animal kingdom.Welch, W. J., et al., The Stress Response and the Immune System,Inflammation: Basic Principles and Clinical Correlates, Raven Press,Gallin, J. I., et al., Eds., Second Edition, 41:841 (1992).

Lymphocyte activation, homing, resistance to target cell lysis, tumorantigenicity, regulation of proto-oncogene transcription, and immunesurveillance are examples of immunologic functions that appear to bemediated or modulated by stress activated signal transduction molecules.Siegelman, M., et al., Science, 231:823 (1986); Kusher, D. I., et al.,J. Immunol., 145:2925 (1990); Ullrich, S. J., et al., PNAS, 83:3121(1986); Colotta, F., et al., Biochem. Biophys. Res. Commun., 168:1013(1990); Haire, R. N., et al., J. Cell Biol, 106:883 (1988); Born, W., etal., Immunol. T., 11:40 (1990). The number of preactivated and MHC classII-restricted autoreactive T-lymphocytes in peripheral blood of patientswith rheumatoid arhritis, for example, dramatically increases relativeto the levels in healthy individuals. Similarly, peripheral bloodT-lymphocytes from patients with inflammatory arthritis proliferatestrongly in the absence of exogenous antigen or mitogen. Welch, W. J.,et al., The Stress Response and the Immune System, Inflammation: BasicPrinciples and Clinical Correlates, Raven Press, Gallin, J. I., et al.,Eds., Second Edition, Chapter 41, 841 (1992). Moreover, synovitis hasbeen shown to result in the generation of oxygen-derived free radicalsthat act to perpetuate tissue damage. Blake, D. R., et al.,Hypoxic-Reperfusion Injury in the Inflamed Human Joint, Lancet, 2:2889(1989).

The control of hematopoiesis is a highly regulated process that respondsto a number of physiological stimuli in the human body. Differentiation,proliferation, growth arrest, or apoptosis of blood cells depends on thepresence of appropriate cytokines and their receptors, as well as thecorresponding cellular signal transduction cascades. Hu, Mickey C. -T.,et al., Genes & Development, 10:2251(1996). Generation of matureleukocytes, for instance, is a highly regulated process which respondsto various environmental and physiological stimuli. Cytokines cause cellproliferation, differentiation or elimination, each of these processesbeing dependent on the presence of appropriate cytokine receptors andthe corresponding signal transduction elements. Moreover, thestimulation of quiescent B- and T-lymphocytes occur via antigenreceptors which exhibit remarkable homology to cytokine receptors.Grunicke, Hans H., Signal Transduction Mechanisms in Cancer,Springer-Verlag (1995).

See also, Suchard, S. J., et al., Mitogen-Activated Protein KinaseActivation During IgG-Dependent Phagocytosis in Human Neutrophils, J.Immunol., 158:4961 (1997).

Distinct signaling cassettes, each containing a central cascade ofkinases, respond to a variety of positive and negative extracellularstimuli, lead to changes in transcription factor activity andposttranslational protein modifications in mammalian cells. Kiefer, F.,et al., EMBO, Vol. 5, 24:7013 (1996). One such protein kinase cascade,known as the mitogen-activated protein kinase (MAPK) cascade, isactivated as an early event in the response of leukocytes to variousstimuli. Stimulation of this pathway has been observed during growthfactor-induced DNA synthesis, differentiation, secretion, andmetabolism. The MAPK pathway has a critical role in the transduction ofreceptor-generated signals from the membrane to the cytoplasm andnucleus. Graves, J. D., et al., Protein Serine/Threonine Kinases of theMAPK Cascade, Annals New York Academy of Sciences, 766:320 (1995). Ithas been established that sustained activation of the MAPK cascade isnot only required, but it is sufficient to trigger the proliferation ofsome cells and the differentiation of others. Cohen, P., Dissection ofProtein Kinase Cascades That Mediate Cellular Response to Cytokines andCellular Stress, Advances in Pharmacology, Academic Press, Hidaka, H.,et al., Eds., Vol. 36, 15 (1996); Marshall, C. J., Cell, 80:179 (1995).Several interdependent biochemical pathways are activated followingeither stimulation of resting T-lymphocytes through the antigen receptoror stimulation of activated T-lymphocytes through the interleukin-2(IL-2) receptor. Many of the events that occur after the engagement ofeither of these receptors are qualitatively similar, such as theactivation of mitogen-activated protein kinase (MAPK) pathways andpreexisting transcription factors, leading to the expression of specificgrowth-associated genes. Symmetry of the Activation of Cyclin-dependentKinaes in Mitogen and Growth Factor-stimulated T Lymphocytes, Jaime F.Modiano, et al., Annals New York Academy of Sciences, 766:134 (1995).

Recent evidence suggests that cellular response to stress is controlledprimarily through events occurring at the plasma membrane, overlappingsignificantly with those important in initiating mitogenic responses.Exposure of cells to biological, chemical, or physical stress agentsevokes a series of events leading to the activation of a wide group ofgenes including transcription factors as well as other gene productsthat are also rapidly and highly induced in response to mitogenicstimulation. The mitogen-activated protein kinase (MAPK) pathway hasbeen shown to be essential for the mitogenic reponse in many systems.See, e.g., Qin, Y. et al., J. Cancer Res. Clin. Oncol., 120:519 (1994).Moreover, due to the fact that most oncogenes encode growth factors,growth factor receptors, or elements of the intracellular postreceptorsignal-transmission machinery, it is becoming increasingly apparent thatgrowth factor signal transduction pathways are subject to an elaboratenetwork of positive and negative cross-regulatory inputs from othertransformation-related pathways. Grunicke, Hans H., Signal TransductionMechanisms in Cancer, Springer-Verlag (1995). The Hierarchicalorganization of the MAPK cascade makes integral protein kinase membersparticularly good targets for such "cross-talk". ProteinSerine/Threonine Kinases of the MAPK Cascade, J. D. Graves, et al.,Annals New York Academy of Sciences, 766:320 (1995).

Initial triggers for inflammation include physical and chemical agents,bacterial and viral infections, as well as exposure to antigens,superantigens or allergens, all of which have the potential to generateReactive Oxygen Species (ROS) and to thereby activate second messengersignal transduction molecules. Storz, G., et al., TranscriptionalRegulators of Oxidative Stress-Inducible Genes in Prokaryotes andEukaryote, in: Stress-Inducible Cellular Responses, Feige, U., et al.,Eds., Birkhauser Verlag (1996). Reactive oxygen radicals, via damage tomany cellular components including DNA, can cause cell death or, if lesssevere, cell cycle arrest at growth-phase checkpoints. Stress damage notonly activates checkpoint controls but also activates protein kinases,including the stress activated protein kinases (SAPKs), c-Raf-1 andERKs, which are integral components of cytoplasmic signal transduction(MAPK) cascades. Pombo, C. M., et al., EMBO, Vol. 15, 17:4537 (1996);Russo, T., et al., J. Biol. Chem., 270:29386 (1995). Considering, interalia, that stress has also been implicated in oxidant injury,atherosclerosis, neurogenerative processes, and aging, elucidation ofthe components of mammalian stress-induced pathways should provide morespecific targets that can be exploited therapeutically. N. J. Holbrook,et al., Stress Inducible Cellular Responses, 273, U. Feige, et al.,Eds., Birkhauser Verlag (1996).

Evidence has demonstrated that mitogen-activated protein kinase (MAPK)and stress activated protein kinase (SAPK) signal transduction pathwaysare responsible for triggering biological effects across a wide varietyof pathophysiological conditions including conditions manifested bydysfunctional leukocytes, T-lymphocytes, acute and chronic inflammatorydisease, auto-immune disorders, rheumatoid arthritis, osteoarthritis,transplant rejection, macrophage regulation, endothelial cellregulation, angiogenesis, atherosclerosis, fibroblasts regulation,pathological fibrosis, asthma, allergic response, ARDS, atheroma,osteoarthritis, heart failure, cancer, diabetes, obeisity, cachexia,Alzheimers disease, sepsis, and neurodegeneration. As MAP kinases play acentral role in signaling events which mediate cellular response tostress, their inactivation is key to the attenuation of the response. N.J. Holbrook, et al., Stress-Inducible Cellular Responses, 273, Feige,U., et al., Eds., Birkhauser Verlag (1996).

Despite major efforts to develop new therapeutic approaches AdultRespiratory Distress Syndrome (ARDS) (acute pulmonary inflammationcharacterized by the massive generation of Reactive Oxygen Species (ROS)within the lung), for example, remains lethal for about 50% of affectedpatients. Polla. B. S., et al., Stress Proteins in Inflammation, in:Stress Inducible Cellular Responses, Feige, U., et al. Eds., BirkhauserVerlag (1996). Moreover, the chronic inflammatory disease, rheumatoidarthritis, for instance, is believed to be mediated by actvatedT-lymphocytes that infiltrate the synovial membrane and initiate aseries of inflammatory processes. Panayi, G. S., et al., The Importanceof the T-Cell in Initiating and Maintaining the Chronic Synovitis ofRheumatoid Arthritis, Arthritis Rheum, 35:729 (1992). Accumulatingevidence also indicates that the autoimmune disease multiple sclerosis(MS) is mediated by autoreactive T-lymphocytes. Stinissen, P., et al.,Crit. Rev. Immunol., 17(1):33 (1997). Autoreactive T-lymphocytes havebeen demonstrated to undergo in vivo activation and clonal expansion inpatients with MS. Zhang, J., et al., J. Mol. Med., 74(11):653 (1996). Indiabetes mellitus, autoreactive T-lymphocytes systematically destroypancreatic islet cells such that they prove incapable of producinginsulin. Another propelling recent development in the implication ofoveractive T-cells is the recognition that a particular subset ofT-lymphocytes appear to be a major culprit in asthma and other allergicdiseases, by responding with undue vigor to apparently harmless invaders(rates of asthma per capita in the developing world have increaseddramatically in the last several decades; doubling in the U.S. since1980). New Clues to Asthma Therapies: Vogel, G., Science, 276:1643(1997).

Recently, much progress has been made in defining the signaltransduction pathways mediating the cellular response to stress. Pombo,C. M., et al., for instance, report the cloning and characterization ofa human Ste20-like oxidant stress response kinase, SOK-1. The kinase ispositively regulated by phohsphorylation and negatively regulated by itsC-terminal non-catalytic region. Reported data suggests SOK-1 transducessignals in response to oxidative and environmental stress. EMBO, Vol.15, 17:4537 (1996). Moreover, Schinkmann, K., et al., recently reportedthe cloning and characterization of the human STE-20-like kinase, mst-3.The mst-3 transcript is reported to be ubiquitously expressed. Mst-1 isfurthermore reported to be positively regulated by autophosphorylation.J. Biol. Chem., 272(45):28695 (1997).Other stress-activated proteinkinase (SAPK), members of the MAPK family, have been shown to beactivated in situ by inflammatory stimuli, including tumor-necrosisfactor (TNF) and interleukin-1. Kyriakis, J. M., et al., Nature, 369:156(1994); Derijard, B., et al., Cell, 76:025 (1994); Sanchez, I., et al.,Nature, 372:794 (1994). See also, Kiefer, F., et al., EMBO, Vol. 5,24:7013 (1996); Creasy, C. L., et al., J. Biol. Chem., 271: No. 35,21049 (1996)); Creasy, C. L., et al., Gene, 167:303 (1995)); Manser, E.,et al., Nature, 367:40 (1994); Hu, Mickey C. -T., et al., Genes &Development, 10:2251(1996); Katz, P., et al., J. Biol. Chem., (1994));Pombo, C. M., et al., Nature, 377:750 (1995).

Integral members of cellular signaling pathways as targets fortherapeutic development, for example, have been the subject to severalreviews. See, e.g., Levitzki, A., Signal-Transduction Therapy: A NovelApproach to Disease Management, Eur. J. Biochem, 226:1 (1994); Powis G.,The Potential for Molecular Oncology to Define New Drug Targets, in: NewMolecular Targets for Cancer Chemotherapy, Workman, P., Kerr D. J.,eds., CRC Press, Boca Raton Fla. (1994). As a result of the efforts ofnumerous laboratories, an impressive list of remarkably specificinhibitors of kinases, for instance, has become available. See, e.g.,Levitzki, A., Tyrphostins: Tyrosine Kinase Blockers as NovelAntiproliferative Agents and Dissectors of Signal Transduction, FASEB;6:3275 (1992); Workman P., et al., Discovery and Design of Inhibitors ofOncogenic Tyrosine Kinases, in: New Approaches in Cancer Pharmacology:Drug Design and Development, Springer, Berlin 55 (1994).

A novel class of pyridinyl imidazoles, CSAIDS [SKB], for instance, havebeen developed, that inhibit the production of the cytokinesinterleukin-1 (IL-1) and tumor necrosis factor (TNF-α) in monocytes. Thedrug has been demonstrated to bind specifically to one protein inmonocytes, termed CSBP (CSAID-binding protein), which has been isolated,cloned, and sequenced and demonstrated as a MAPK homolog. Lee, J. C., etal., Differential Effects of the BicyclicImidazoles on CytokineSynthesis in Human Monocytes and Endothelial Cells, Agents Actions,41:C191 (1994); A Protein Kinase Involved in the Regulation ofInflammatory Cytokine Biosynthesis, Nature, 372:739 (1994). Moreover, asdemonstrated by the identification of rapamycin as a specific inhibitorof the activation of p70 S6 kinase and the identification of compoundsthat inhibit the EGF receptor protein kinase very potently and thatblock the activation of MAP kinase kinase have demonstrated thatspecific inhibitors of protein kinases can indeed be developed. Alessi,D., et al., A Specific Inhibitor of the Activation of MAP KinaseKinase-1 in vitro and in vivo, J. Biol. Chem., 279:27489 (1995); Fry,D., et al., A Specific Inhibitor of the Epidermal Growth Factor ReceptorTyrosine Kinase, Science, 265:806 (1994); Cohen, P., Dissection ofProtein Kinase Cascades That Mediate Cellular Response to Cytokines andCellular Stress, Intracellular Signal Transduction, Advances inPharmacology, Hidaka, H., et al., Eds., Academic Press, 36:17 (1996).

Compounds which are able to modulate the activity of specific signaltransduction molecules integral to specific intracellular pathways areexpected to have significant potential for the ability to control orattenuate downstream physiological responses. Unfortunately, in spite ofthe introduction of numerous new drugs during the last three decades,there is a need for new, more efficient and less toxic compounds.Accordingly, the ability to identify such compounds is of paramountimportance.

SUMMARY OF THE INVENTION

The present invention is directed to an isolated and purifiedpolynucleotide molecule comprising a nucleic acid sequence which encodesa polypeptide comprising an amino acid sequence having at least about90% homology to a member selected from the group consisting of: (SEQ IDNO:3, SEQ ID NO:3 positions 24-274, and SEQ ID NO:3 positions 275-416).

The current invention is directed to an antisense molecule comprising anoligomer in the range from about 12 to about 25 nucleotides in lengthwhich: (a) is complementary to a region within positions 157-232 orpositions 1405-1480 of SEQ ID NO:1.

The invention is further directed to a method of identifying compoundsthat modulate a biological and/or pharmacological activity of a signaltransduction kinase polypeptide, comprising combining a candidatecompound modulator with a polypeptide comprising an amino acid sequencehaving at least about 90% homology to a member selected from the groupconsisting of: (SEQ ID NO:3, SEQ ID NO:3 positions 24-274, and SEQ IDNO:3 positions 275-416), and measuring an effect of the candidatecompound modulator on the biological and/or pharmacological activity ofthe polypeptide.

The invention is further directed to a method of modulating a biologicaland/or pharmacological activity of a signal transduction kinasepolypeptide in a cell comprising administering an effective amount of apolynucleotide--or--an effective amount of an antisense molecule to saidcell.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All publications and patentsreferred to herein are incorporated by reference.

Biological activity as used herein in reference to the signaltransduction serine threonine protein kinase of the present inventionrefers to the ability of the biomolecule to transduce cellular signalsincluding but not limited to the ability to bind ATP, toautophosphorylate and/or to phosphorylate a substrate and to the abilityto directly or otherwise activate any of the biological moleculesincluding but not limited to NFκB, AP-1, and SRF.

Pharmacological activity as used herein in reference to the signaltransduction serine threonine protein kinase of the present inventionrefers to the ability to mediate any one or more of the physiologicalconditions including but not limited to cell differentiation,proliferation, oncogenic transformation, macrophage regulation,endothelial cell regulation, fibroblasts regulation, cytoskeletalstructure, metastases, cell aggregation, cell motility, cytokinesis,acute and chronic inflammatory disease, auto-immune disorders, allergicresponse, secretion, apoptosis, neurological disorders, peripheralvascular disease, atherosclerosis, and asthma.

Dominant negative mutant as used herein refers to a nucleic acid codingregion sequence which has been changed with regard to at least oneposition in the sequence, relative to the corresponding wild type nativeversion, preferrably at a position which encodes a changed amino acidresidue position at an active site required for biological and/orpharmacological activity in the native peptide. Dominant negative mutantembodiments of the invention, for example, include peptides comprising asequence as depicted in SEQ ID NO:3, SEQ ID NO:3 positions 24-274, orSEQ ID NO:3 positions 275-416 wherein one or more positionscorresponding to SEQ ID NO:3 selected from the group consistingessentially of (position 31 (glycine), 33 (glycine), 36 (glycine), 38(valine), 51 (alanine), 53 (lysine), 144 (aspartic acid), 149(asparagine), 162 (aspartic acid), 163 (phenylalanine), 164 (glycine),182 (threonine), 189 (glutamic acid), and 201 (aspartic acid)) aresubstituted or deleted. Dominant negative mutants are moreover definedto be included within the scope of the disclosure of the variantssection infra.

Biologically effective as used herein in reference to antisense nucleicacid molecules as well as dominant negative mutant nucleic acid codingregions and dominant negative mutant peptides refers to the ability ofthese molecules to modulate the biological activity and/orpharmacological activity of the novel signal transduction protein kinaseof the present invention and/or transcription/translation of nucleicacid coding regions of the novel signal transduction protein kinase ofthe present invention.

As used herein, a functional derivative of a biomolecule disclosedherein is an entity that possesses a functional biological activityand/or pharmacological activity as defined herein that is derived fromSEQ ID NO:1 or SEQ ID NO:3, for example, truncated versions, versionshaving deletions, functional fragments, versions having substitutions,versions having insertions or extended ends, or biologically effectivedominant negative mutants as well as biologically effective antisensemolecules.

The term modulation is used herein to refer to the capacity to eitherenhance or inhibit a biological activity and/or pharmacological activityof a signal transduction molecule of the present invention or to thecapacity to either enhance or inhibit a functional property of a nucleicacid coding region of the present invention. Modulate physiology as usedherein refers to the biophysiological regulation of cells and/or tissueand the treatment of pathophysiological disorders related thereto.

Direct administration as used herein refers to the direct administrationof nucleic acid molecules, peptides, or compounds which compriseembodiments and/or functional derivatives (e.g., SEQ ID NO:1, SEQ IDNO:3) of the present invention. Direct administration includes but isnot limited to gene therapy.

Purified as used herein refers to molecules, either nucleic acid oramino acid sequences, that are removed from their natural environmentand isolated or separated from at least one other component with whichthey are naturally associated.

Expression vector as used herein refers to nucleic acid vectorconstructions to direct the transcription of nucleic acid regions inhost cells. Expression vectors include but are not limited to plasmids,retroviral vectors, viral and synthetic vectors.

Transformed host cells as used herein refer to cells which harbornucleic acids or functional derivatives of the present invention.

signal transduction via kinases

A cascade signal transduction mechanism is essentially a conduit for thetransmittal of an external stimulus to the cell nucleus in order totrigger a distinct pattern of gene expression in response to thestimulation. The extracellular signal is amplified and transduced by aseries of independent sequential covalent modifications, from the plasmamembrane to the nucleus, via distinct phosphorylation steps ofindependently specific cognate cytosolic biomolecules. Proteinphosphorylation is now acknowledged as the most important means of acuteregulation of protein function, signal transduction, and gene expressionin eukaryotic cells. Intracellular biomolecule phosphorylation viaspecific kinases is responsible for switching of cellular activity fromone state to another. It is the major mechanism by which cells respondto extracellular signals such as mitogenic stimulation; biological,chemical, and physical stress, hormones, and growth factors. ProteinPhosphorylation, Hardie, D. G., Oxford Press (1993).

The protein kinases are a large family of enzymes. Conserved structuralmotifs provide clear indications as to how the kinases transfer theγ-phosphate of a purine nucleotide triphosphate to the hydroxyl groupsof their protein substrates. There are two main subdivisions within thesuperfamily: the protein-serine/threonine kinases and theprotein-tyrosine kinases. The kinase domains that define protein kinasescontain 12 conserved subdomains (I-XII) that fold into a commoncatalytic core structure, as revealed by the 3-dimensional structures ofseveral enzymes. The central core of the catalytic domain, the regionwith greatest frequency of highly conserved residues, consists ofsubdomains VI through IX. The most striking indicator of amino acidspecificity is found in subdomain VI, the consensus in this region is astrong indicator of serine/threonine specificity. See, e.g., Hanks, S.K., et al., The Protein Kinase Family: Conserved Features and DeducedPhylogeny of the Catalytic Domains, Science, 241:42 (1988); Hanks, S.K., et al., The Eukaryotic Protein Kinase Superfamily: Kinase(Catalytic) Domain Structure and Classification, FASEB , Ser. Rev.,9:576 (1995).

Protein kinases which have closely related catalytic domains, and thusdefine a family, represent products of genes that have undergonerelatively recent evolutionary divergence. Clustering appears to be ofpredictive value in the determination of the properties and function ofnovel protein kinases. Accordingly, members of a given family tend alsoto share related functions. This is manifest by similarities in overallstructural topology, mode of regulation, and substrate specificity. See,generally, Hardie, D. G,. et al., The Protein Kinase Factsbook, AcademicPress, London (1995).

Progress has been made by many labs in defining signaling pathwaysinitiated by mitogenic stimuli. Blenis, J., Signal Transduction via theMAP Kinases, PNAS, 90:5889 (1993). The MAP kinase family of enzymes havebeen implicated as common and essential components of signaling pathwaysinduced by diverse mitogenic stimuli. Once activated, MAP kinasesphosphorylate a number of substrates including transcription factorsessential for triggering gene expression required for the growthresponse. Accordingly, the MAP kinases are considered to be potentiallyvaluable pharmacological targets within the growth factor signalingpathways. Hidaka, H., et al., Intracellular Signal Transduction,Advances in Pharmacology, Academic Press (1996).

Mitogen-activated protein kinases (MAPKs) and their upstream regulatorykinases comprise functional units that couple upstream input signals toa variety of outputs. MAPK cascades have been remarkably conserved inevolution. The core of these cascades is a three-tiered moduleconsisting of an MAPK-extracellular signal-regulated kinase kinase (anMEKK), an MEK and an MAPK or extracellular signal-regulated kinase(ERK). The defining characteristic of these modules is the MAPK itself.The classical pathway, known as the extracellular signal-regulatedkinase pathway (ERK), is activated by mitogens and growth factors. ERKhas a regulatory kinase, MAPK kinase or MEK, necessary for activation.This enzyme is in turn regulated by another MAPKK kinase known as Raf.Analogous with the classical MAPK module are two other modules which areactivated by cytokines and cellular stresses and which have become knownas the stress kinase pathways. The defining MAPKs of these pathways areJNK (SAPK) and P38. JNK is activated by the upstream SEK-1 (MKK4) whichis activated by MEKK1 or MLK3 whereas P38 is activated by MKK3 and MKK6.

In yeast the MAPK modules operate in a linear manner linkingextracellular signals to functional response, whereas in mammalian cells`cross-talk` between modules may be obligatory in some cases (eg. IL-2production by T-lymphocytes). Phosphorylation of transcriptional factors(eg. AP-1, NF-kB, ELK-1, ATF-2) by the terminal MAPKs serve to regulateexpression of key inflammatory genes. Differences in the expression ofthe various kinases between cell types and in response to processes indisease will have major impact on how cells respond to extracellularstimuli under physiological and pathological conditions. It is for thesereasons that there is very likely to be selectivity for specificinhibitors of these different kinases for their associated physiologicalrole as well as opportunities for therapeutic intervention.

Phosphorylation of transcriptional factors (eg. AP-1, NF-kB, ELK-1,ATF-2) by the terminal MAPKs serve to regulate expression of keyinflammatory genes. Differences in the expression of the various kinasesbetween cell types in response to processes in disease will have majorimpact on how cells respond to extracellular stimuli under physiologicaland pathological conditions. This should provide opportunities fortherapeutic intervention.

Studies of the budding and fission yeasts, Saccharomyces cerevisiae andSchizosaccharomyces pombe, have been particularly fruitful in therecognition of protein kinases. Hanks, S. K., et al., The EukaryoticProtein Kinase Superfamily, FASEB Ser. Rev., 9:576 (1995). Signaltransduction pathways connecting cell surface receptors with each memberof the MAPK superfamily in mammalian cells are remarkably similar tothose of the budding yeast Saccharamyces cerevisiae, in which geneticstudies have shown parallel signaling cascades leading to the activationof at least three distinct MAPK-related kinases. Hu, Mickey C. -T., etal., Genes & Development, 10:2251(1996); See, e.g., Herskowitcz, I., etal., Cell, 80:187 (1995).

A component of the pheromone-response pathway in budding yeast, Ste20,represented the first identified member of a new family ofserine/threonine protein kinases. Leberer, E., et al., EMBO, 11:4815(1992); Ramer, S. W., et al., PNAS, 90:452 (1993). Several mammalianhomologs to Ste20 have since been identified, including MST1 (Creasy, C.L., et al., J. Biol. Chem., 271: No. 35, 21049 (1996)), MST2 (Gene,167:303 (1995)), HPK1 (Kiefer, F., et al., EMBO, Vol. 5, 24:7013(1996)). Recently, mammalian Ste20-like kinases, including p21-activatedprotein kinase (PAK1) (Manser, E., et al., Nature, 367:40 (1994)) andgerminal center kinase (GC kinase) (Katz, P., et al., J. Biol. Chem.,(1994)), have been shown to be capable of activating mammalian MAPKcascades. Pombo, C. M., et al., EMBO, Vol. 15, 17:4537 (1996). See also,U.S. Pat. No. 5,605,825, Human PAK65, issued Feb. 25, 1997. Methodsdescribed in U.S. Pat. No. 5,605,825 are herein incorporated herein byreference.

The MAP kinases require activation by a MAPK/ERK activating kinase(MEK). The dual-specificity kinase is capable of phosphorylating bothtyrosine and serine/threonine residues in proteins. The proto-oncogenec-Raf-1, for instance, has been shown to encode a protein acting as aMEK kinase and the pathway Raf→MEK→MAPK is now well established as amajor signal transduction pathway for growth factors. Activated MAPKundergoes a translocation to the nucleus where it can directlyphosphorylate and activate a variety of transcription factors includingc-Myc, C/EBPβ, p62^(TCF) /Elk-1, ATF-2 and c-Jun. Grunicke, Hans H.,Signal Transduction Mechanisms in Cancer, Springer-Verlag (1995).

The MAP kinase (MAPK) cascade is critical in mediating severalintracellular actions. In one pathway a series of protein-proteininteractions is triggered at the plasma membrane that culminate inactivation of the GTP-binding protein Ras. GTP-Ras then interacts withthe protein kinase Raf, recruiting it to the plasma membrane where it isactivated. The activation of Raf is followed by the sequentialactivation of three additional kinases: MAP kinase kinase (MAPKK), MAPK,and MAPK-activated protein (MAPKAP) kinase-1. The activation of MAPK andMAPKAP kinase-1 leads to their translocation from the cytosol to thenucleus where they regulate the activities of transcription factors.Cohen, P., Dissection of Protein Kinase Cascades That Mediate CellularResponse to Cytokines and Cellular Stress, Advances in Pharmacology,Academic Press, Hidaka, H., et al., Eds., Vol. 36, 15 (1996); Marshall,C. J., Cell, 80:179 (1995).

Recent evidence suggests that cellular response to stress is controlledprimarily through events occurring at the plasma membrane, overlappingsignificantly with those important in initiating mitogenic responses.Exposure of cells to stress agents, as mentioned supra, evokes a seriesof events leading to the activation of a wide group of genes includingtranscription factors as well as other gene products that are alsorapidly and highly induced in response to mitogenic stimulation.Pathways which are involved in mediating these cellular responses relyon the activation of mitogen-activated protein kinases (MAPK) whichinclude extracellular signal-regulated kinases (ERK), stress activatedprotein kinases (SAPK), c-Jun N-terminal kinases (JNK), and p38/PK/CSBPkinases. These kinases play a key role in the activation oftranscription factors and other regulatory proteins involved inactivating gene expression. Phosphorylation enhances complex formationand the serum response element located in the promoters of stressresponse genes such as c-fos. Other regulated proteins includep90^(RSK), activating transcription factor-2 (ATF-2, a cAMP responseelement-binding protein), NF-IL6 (nuclear factor for the activation ofInterleukin 6) and c-Myc. Davis, R. J., The Mitogen-Activated ProteinKinase Signal Transduction Pathway, J. Biol. Chem., 268:1553 (1993); N.J. Holbrook, et al., Stress Inducible Cellular Responses, 273, in:Stress-Inducible Cellular Responses, Feige, U., et al., Eds., BirkhauserVerlag (1996). The mitogen-activated protein kinase (MAPK) pathway hasbeen shown to be essential for the mitogenic reponse in many systems.See, e.g., Qin, Y. et al., J. Cancer Res. Clin. Oncol., 120:519 (1994).Moreover, due to the fact that most oncogenes encode growth factors,growth factor receptors, or elements of the intracellular postreceptorsignal-transmission machinery, it is becoming increasingly apparent thatgrowth factor signal transduction pathways are subject to an elaboratenetwork of positive and negative cross-regulatory inputs from othertransformation-related pathways. Grunicke, Hans H., Signal TransductionMechanisms in Cancer, Springer-Verlag (1995). The Hierarchicalorganization of the MAPK cascade makes integral protein kinase membersparticularly good targets for such "cross-talk". ProteinSerine/Threonine Kinases of the MAPK Cascade, J. D. Graves, et al.,Annals New York Academy of Sciences, 766:320 (1995).

Initial triggers for inflammation include physical and chemical agents,bacterial and viral infections, as well as exposure to antigens,superantigens or allergens, all of which have the potential to generateReactive Oxygen Species (ROS) and to thereby activate second messengersignal transduction molecules. Reactive oxygen species are generatedfrom molecular oxygen and include the free radicals superoxide (·O₂ ⁻),hydroxyl (·OH) and nitric oxide (NO·), as well as non-radicalintermediates such as hydrogen peroxide (H₂ O₂) and singlet oxygen (¹O₂). During normal cellular respiration, ROS are constantly produced atlow rate in both eukaryotes and prokaryotes. At these lowconcentrations, ROS can act as second messengers, stimulate cellproliferation, and act as mediators for cell activation. However, duringphagocytosis, infection or inflammation, ROS can accumulate to toxiclevels which leads to oxidative stress, and may damage almost allcellular components. All organisms have mechanisms to detoxify theoxidants or to repair the damage caused by ROS, including superoxidedismutases, catalases, peroxidases, glutathione, thioredoxin, and heatshock proteins. The expression of the genes coding these proteins(oxidative stress genes) is induced by changes in the concentrations ofROS, suggesting that cells have developed mechanisms to sense the ROS.Storz, G., et al., Transcriptional Regulators of OxidativeStress-Inducible Genes in Prokaryotes and Eukaryote, in:Stress-Inducible Cellular Responses, Feige, U., et al., Eds., BirkhauserVerlag (1996).

Sok-1

Pombo et al. recently reported the cloning and characterization of humanSte20/oxidant stress response kinase, SOK-1 which belongs to the Sps1/GCkinase group of Ste20-like kinases with N-terminal catalytic domains.The kinase is positively regulated by phohsphorylation and negativelyregulated by its C-terminal non-catalytic region. SOK-1 is activatedrelatively specifically by oxidant stress. Reported data places SOK-1 ina stress response pathway and suggest it resembles in function yeastSte20s which transduce signals in response to environmental stress.EMBO, Vol. 15, 17:4537 (1996). The open reading frame is reported toencode a protein of 426 amino acids and has a predicted M_(r) of 48,041Da. The kinase domain is located in the N-terminal half of the protein.The reported peptide contains all 11 subdomains of serine/threoninekinases. Ste20 related stress-activated kinases are evidenced to be inproximity to the membrane in the signaling cascade and therefore areable to provide greater target opportunity for selective modulation ofsignal transduction.

Mst-3

Schinkmann et al. recently reported the cloning and characterization ofthe human STE-20-like kinase, mst-3. The mst-3 transcript is reported tobe ubiquitously expressed, is most closely related to SOK-1, and isfurthermore reported to be positively regulated by autophosphorylation.A cDNA molecule composed of 1976 bases has been described as well as anopen reading frame which encodes a 431 amino acid peptide (SEQ ID NO:5)having a molecular mass of 48 kDa. The mst-3 kinase is reported toconsist of an N-terminal kinase domain and a 142-amino acid C-terminalregulatory domain. The mst-3 molecule shares 68.8% overall amino acidhomology with SOK-1. J. Biol. Chem., 272(45):28695 (1997).

novel human signal transduction serine/threonine kinase

A novel human signal transduction serine/threonine protein kinasemolecule, as well as example nucleic acid sequences which encodetherefor, are herein described.

A cDNA sequence is provided, SEQ ID NO:1, which comprises the structuralcoding region of the native human signal transduction kinase, SEQ IDNO:2. The 3201 bp SEQ ID NO:1 contiguous cDNA sequence contains a 1251bp open reading frame (ORF) with a Kozak initiation sequence at thestart methionine. SEQ ID NO:2 shows the 1251 base open reading frameincluding the stop codon. The native human homolog of the Ste20-likesignal transduction kinase is shown in SEQ ID NO:3. The 416 amino acidresidue sequence of the novel protein contains all eleven (11) sudomainsfound in eukaryotic protein kinases including Ste20-like kinases. SEQ IDNO:3 has 65.7 and 71 percent homology to the Ste20/oxidant stressresponse kinse-1 (SOK-1), SEQ ID NO:4, described by Pombo et al., and tothe mammalian Ste20-like kinase (MST-3), SEQ ID NO:5, described bySchinkmann et al., respectively. EMBO, Vol. 15, 17:4537 (1996); J. Biol.Chem., 272(45):28695 (1997). The novel kinase has a predicted molecularweight of 46527.83 daltons, an isoelectric point of 5.097, and a netcharge of -13 at pH 7.0.

Mitogen-activated protein kinase cascades have been remarkably conservedin evolution. SEQ ID NO:4, for example, shows the 426 amino acid residuesequence of SOK-1 which is the recently described human MAPK-pathwayoxidant (stress) activated kinase. SOK-1 has been characterized as ahuman homolog of the MAPK-pathway yeast stress-activated kinase Ste20.Pombo, C. M., et al., EMBO, Vol. 15, 17:4537 (1996). Pombo et al.reported SEQ ID NO:4 as a human Ste20/oxidant stress response kinase.Ste20 related stress-activated kinases, via evidentiarycharacterization, appear to be close to the plasma membrane in thesignaling cascade and therefore may have significant potential toprovide greater target opportunity for selective modulation of signaltransduction. EMBO, Vol. 15, 17:4537 (1996).

Some features of the cDNA SEQ ID NO:1 are for instance: bases 1-181represent the 5' UTR, bases 176-185 represent the Kozak sequence, bases182-1432 represent CDS, bases 1433-3201 represent 3' UTR, bases3194-3199 represent the poly adenylation signal.

Some features of the 416 residue polypeptide (SEQ ID NO:3) are forinstance: Molecular Weight 46527.83 Daltons, 51 Strongly Basic(+) AminoAcids (K,R), 65 Strongly Acidic(-) Amino Acids (D,E), 138 HydrophobicAmino Acids (A,I,L,F,W,V), 103 Polar Amino Acids (N,C,Q,S,T,Y),Isolectric Point=5.097, Charge at PH 7.0=-13.017.

Eukaryotic protein kinases each contain a conserved catalytic domainregion of 250-300 amino acid residues which is responsible for thephosphotransferase activity of the enzyme. The catalytic domain of thenovel signal transduction kinase described herein (SEQ ID NO:3) has 89and 87.5 percent similarity, respectively, with the catalytic domain ofmst-3 (SEQ ID NO:5) and SOK-1 (SEQ ID NO:4). Catalytic domains can befurther divided into 12 subdomains defined by strings of invariant andconserved residues. Residue positions 31-38 of the novel signaltransduction kinase, for instance, correspond to the consensus kinasesubdomain I GxGxxGxV. Subdomain II is involved in the phosphotransferreaction and identified by an invariant lysine in the tripeptidesequence AxK. With regard to the novel kinase SEQ ID NO:3, subdomain IIis found in residues 51-53 (AIK). Subdomains VI through IX,characterized by a large number of highly conserved residues, form thecentral core of catalytic activity. SEQ ID NO:3 comprises the invariantor nearly invariant residues Asp¹⁴⁴ and Asn¹⁴⁹ in subdomain VI andAsp¹⁶², Phe¹⁶³, and Gly¹¹⁶⁴ in subdomain VII; all of which have beenimplicated in ATP binding. Region VIB contains the consensus sequenceHRDLxxxN, with D being the invariant Asp¹⁴⁴. SEQ ID NO:3 in this regionis HRDIKAAN (wherein the substitution of Ile for Leu is conservative).Moreover, the novel signal transduction kinase contains the conservedDFG of subdomain VII. The Asp functions to orient the γ-phosphate of theATP for transfer. Subdomain VIII of SEQ ID NO:3 contains the highlyconserved APE sequence, with the Glu corresponding to the invariantGlu¹⁸⁹. This subdomain is thought to play a critical role in therecognition of substrate binding. Additionally, many kinases are knownto be activated by phosphorylation of residues in subdomain VIII. Thesequence DxWS/AxG of subdomain IX is represented by amino acid positions201-206 of SEQ ID NO:3 (DIWSLG). This region forms a large α-helix andthe initial Asp of the consensus sequence serves to stabilize thecatalytic loop by hydrogen bonding.

SEQ ID NO:3 is capable of autophosphorylation and substratephosphorylation (e.g., myelin basic protein). SEQ ID NO:3 positions24-274 represent the catalytic domain of the serine threonine signaltransduction kinase. SEQ ID NO:3 positions 275-416 represent theregulatory region. Therefore, particularly preferred embodiments of thecurrent invention are polynucleotides which comprise a nucleic acidsequence which encodes a polypeptide comprising an amino acid sequenceof SEQ ID NO:3 positions 24-274, or SEQ ID NO:3 positions 275-416.Polypeptides are preferred which comprise SEQ ID NO:3 amino acidpositions 24-274 and/or SEQ ID NO:3 positions 275-416.

expression in immune tissues

Northern blot analysis identifies a primary transcript pertaining to thenovel signal transduction kinase of approximately 3.5 kb in length.Prominent transcripts are apparent primarily in immune tissues(expressed at a particularly high level in tissues of the human immunesystem, i.e., lymph node, peripheral blood leukocytes, spleen, fetalliver, bone marrow, thymus, and placenta) as well as from placenta andcancer-cell lines. See, EXAMPLE V. Expression of the novel kinaseappears to be in sharp contrast to the mst-3 transcript which isreported to be ubiquitously expressed. J. Biol. Chem., 272(45):28695(1997).

activation of transcription factors by TEN-1 kinase

The signal transduction kinase (SEQ ID NO:3) is demonstrated herein toactivate NFκB, AP1, and SRF when overexpressed in cell lines (e.g.,HEK293, HeLa). In addition, expression of an antisense cDNA has beenshown to inhibit both basal and TNFa-mediated activation of NFκB inECV304 cells. Shown in Example XI is the general format of thetranscription activation reporter assays used to determine if SEQ IDNO:3 activates specific transfactors. The PathDetect™ System (includingreporter constructs and positive control constructs) was used to performthese assays. Stratagene, La Jolla, Calif. All assays were performed intriplicate and repeated several separate times.

The signal transduction kinase (SEQ ID NO:3) activates NFκB, AP1, andSRF when overexpressed in HEK293 and HeLa cells. In addition, SEQ IDNO:1 anti-sense cDNA expression in ECV304 cells is capable of inhibitingboth basal and TNFa stimulated inductions of NFκB by approximately 66%and 50%, respectively. SEQ ID NO:3 is able to activate transcriptionfactors (e.g. NFκB, API) in vivo that are known to initiatetranscription of numerous inflammatory genes including pro-inflammatorycytokines, chemokines, inflammatory enzymes, adhesion molecules, andinflammatory receptors. In addition, NFκB synergistically interacts withAP 1 to promote gene transcription. The co-operativity between NFκB andother transcription factors activated by inflammatory signals underliethe specificity of the inflammatory response in many different diseases.

NFκB, AP-1, and SRF are activated in 293 cells by the signaltransduction kinase SEQ ID NO:3. MEKK is a known activator of NFκB andis supplied as part of the Stratagene PathDetect™ System. Moreover,AP-1, and SRF are abctivated in HeLa cells by the signal transductionkinase SEQ ID NO:3. MEKK is a known activator of AP-1 and is supplied aspart of the Stratagene PathDetect™ System. PKA (protein kinase A) is aknown activator of SRF and is supplied as part of the StratagenePathDetect™ System. SEQ ID NO:1 antisense molecules greatly reduce thebasal level and TNF α-mediated NFκB activation in ECV304 cells.

example dominant negative mutants

A preferred polynucleotide of the present invention comprises a nucleicacid sequence which encodes a polypeptide comprising the sequence asdepicted in SEQ ID NO:3 wherein one or more positions of SEQ ID NO:3selected from the group consisting essentially of (position 31(glycine), 33 (glycine), 36 (glycine), 38 (valine), 51 (alanine), 53(lysine), 144 (aspartic acid), 149 (asparagine), 162 (aspartic acid),163 (phenylalanine), 164 (glycine), 182 (threonine), 189 (glutamicacid), and 201 (aspartic acid)) are substituted or deleted. See, forinstance, Example X.

variants

The present invention relates to nucleic acid (e.g., SEQ ID NO:1 and SEQID NO:2) and amino acid sequences (e.g., SEQ ID NO:3) of the novel humansignal transduction kinase and variations thereof as well as functionalderivatives and to the use of these sequences to identify compounds thatmodulate the biological and/or pharmacological activity of the signaltransduction molecule. Further, the invention relates to biologicallyeffective antisense nucleic acid molecules as well as nucleic acidswhich encode biologically effective dominant negative mutant versions ofthe signal transduction kinase as well as biologically effectivedominant negative mutant versions of the novel peptide biomolecule. Thepresent invention also provides a method of treatment for a patient inneed of such treatment, videlicet for a patient who suffers apathological condition mediated by the SEQ ID NO:3 signal transductionkinase, comprising administering an effective amount of a biologicallyeffective antisense nucleic acid molecule derived from SEQ ID NO:1; oradministering an effective amount of a nucleic acid which encodes abiologically effective dominant negative mutant version of the signaltransduction kinase; or administering a compound that modulates thebiological and/or pharmacological activity of SEQ ID NO:3 which wasidentified by a method described herein.

The present invention also encompasses variants of the humansignal-transduction kinase molecule SEQ ID NO:3. A preferred variant,for instance, is a polypeptide comprising a fragment having at least 90%amino acid sequence homology (identity) to SEQ ID NO:3; a more preferredvariant is one having at least 93% amino acid sequence homology; and amost preferred variant is one having at least 95% amino acid sequencehomology to the signal-transduction kinase amino acid sequence asdepicted in SEQ ID NO:3 or a biologically and/or pharmacologicallyactive substantial fragment thereof, e.g., SEQ ID NO:3 positions 24-274,and SEQ ID NO:3 positions 275-416. Variants within the scope of thisinvention also include dominant negative mutants of these contemplatedembodiments, examples of which are set forth herein.

A variant of the SEQ ID NO:3 signal-transduction kinase molecule of thepresent invention may have an amino acid sequence that is different byone or more amino acid substitutions. Embodiments which comprise aminoacid deletions and/or additions are also contemplated. The variant mayhave conservative changes (amino acid similarity), wherein a substitutedamino acid has similar structural or chemical properties, for example,the replacement of leucine with isoleucine. A variant may havenonconservative changes, e.g., replacement of a glycine with atryptophan. Embodiments within the intended scope of the invention alsoinclude SEQ ID NO:3 having one or more amino acid deletions orinsertions, or both. Guidance in determining which and how many aminoacid residues may be substituted, inserted or deleted without abolishingbiological or proposed pharmacological activity may be reasonablyinferred in view of this disclosure and may be further be found usingcomputer programs well known in the art, for example, DNAStar software.

Amino acid substitutions of SEQ ID NO:3 may be made, for instance, onthe basis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asa biological and/or pharmacological activity of the native molecule isretained. However, amino acid substitutions are important to constructcontemplated biologically effective dominant negative mutants, severalspecies of which are set forth herein.

Negatively charged amino acids, for example, include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine;glycine, alanine; asparagine, glutamine; serine, threoninephenylalanine, and tyrosine. However, in the construction ofbiologically effective dominant negative mutants at least one amino acidresidue position at an active site required for biological and/orpharmacological activity in the native peptide is changed to produce anagent or entity having reduced activity or which is devoid of detectablenative wild type activity.

Suitable substitutions of amino acids include the use of a chemicallyderivatized residue in place of a non-derivatized residue. D-isomers aswell as other known derivatives may also be substituted for thenaturally occurring amino acids. See, e.g., U.S. Pat. No. 5,652,369,Amino Acid Derivatives, issued Jul. 29, 1997. Example substitutions areset forth in TABLE 1 as follows:

                  TABLE 1                                                         ______________________________________                                        Original residue                                                                             Example conservative substitutions                             ______________________________________                                        Ala (A)        Gly; Ser; Val; Leu; Ile; Pro                                     Arg (R) Lys; His; Gln; Asn                                                    Asn (N) Gln; His; Lys; Arg                                                    Asp (D) Glu                                                                   Cys (C) Ser                                                                   Gln (Q) Asn                                                                   Glu (E) Asp                                                                   Gly (G) Ala; Pro                                                              His (H) Asn; Gln; Arg; Lys                                                    Ile (I) Leu; Val; Met; Ala; Phe                                               Leu (L) Ile; Val; Met; Ala; Phe                                               Lys (K) Arg; Gln; His; Asn                                                    Met (M) Leu; Tyr; Ile; Phe                                                    Phe (F) Met; Leu; Tyr; Val; Ile; Ala                                          Pro (P) Ala; Gly                                                              Ser (S) Thr                                                                   Thr (T) Ser                                                                   Trp (W) Tyr; Phe                                                              Tyr (Y) Trp; Phe; Thr; Ser                                                    Val (V) Ile; Leu; Met; Phe; Ala                                             ______________________________________                                    

"Homology" is a measure of the identity of nucleotide sequences or aminoacid sequences. In order to characterize the homology, subject sequencesare aligned so that the highest order homology (match) is obtained."Identity" per se has an art-recognized meaning and can be calculatedusing published techniques. Computer program methods to determineidentity between two sequences, for example, include DNAStar software(DNAStar Inc., Madison, Wis.); the GCG program package (Devereux, J., etal., Nucleic Acids Research (1984) 12(1):387); BLASTP, BLASTN, FASTA(Atschul, S. F. et al., J Molec Biol (1990) 215:403). Homology(identity) as defined herein is determined conventionally using the wellknown computer program, BESTFIT (Wisconsin Sequence Analysis Package,Version 8 for Unix, Genetics Computer Group, University Research Park,575 Science Drive, Madison, Wis. 53711). When using BESTFIT or any othersequence alignment program to determine whether a particular sequenceis, for example, about 90% homologous to a reference sequence, accordingto the present invention, the parameters are set such that thepercentage of identity is calculated over the full length of thereference nucleotide sequence or amino acid sequence and that gaps inhomology of up to about 10% of the total number of nucleotides in thereference sequence are allowed. Ninety percent of homology is thereforedetermined, for example, using the BESTFIT program with parameters setsuch that the percentage of identity is calculated over the full lengthof the reference sequence, e.g., SEQ ID NO:3, and gaps of up to 10% ofthe total number of amino acids in the reference sequence are allowed,and wherein up to 10% of the amino acid residues in the referencesequence may be deleted or substituted with another amino acid, or anumber of amino acids up to 10% of the total amino acid residues in thereference sequence may be inserted into the reference sequence. Percenthomologies are likewise determined, for example, to identify preferredspecies, within the scope of the claims appended hereto, which residewithin the range of about 90 percent to 100 percent homology to SEQ IDNO:3 as well as biologically and/or pharmacologically active functionalderivatives thereof and biologically effective dominant negative mutantscontemplated herein.

Percentage similarity (conservative substitutions) between twopolypeptides may also be scored by comparing the amino acid sequences ofthe two polypeptides by using programs well known in the art, includingthe BESTFIT program, by employing default settings for determiningsimilarity.

The present invention relates to nucleic acid sequences and amino acidsequences of the signal-transduction kinase and variants thereof and tothe use of these sequences to identify compounds that modulate thebiological and/or pharmacological activity of a signal transductionmolecule.

Polynucleotide sequences which encode the signal-transduction kinase asdepicted in SEQ ID NO:3 and variants thereof contemplated herein areparticularly preferred embodiment of the present invention. Biologicallyeffective antisense molecules and nucleic acids which encodebiologically effective dominant negative mutant versions of SEQ ID NO:3,or derivatives thereof, as well as dominant negative mutant versions ofSEQ ID NO:3, and derivatives thereof, examples of each of which aredescribed infra, are preferred embodiments of the present invention andare intended to fall within the scope of the claims appended hereto.

The present invention relates to nucleic acid sequences (e.g., SEQ IDNO:1 and SEQ ID NO:2) and amino acid sequences (e.g., SEQ ID NO:3, SEQID NO:3 positions 1-86, SEQ ID NO:3 positions 87-124, SEQ ID NO:3positions 125-155, SEQ ID NO:3 positions 248-266, SEQ ID NO:3 positions266-288, and SEQ ID NO:3 positions 289-298) of the novel human signaltransduction kinase as well as inherent derivatives thereof, e.g.,functional derivative that demonstrate or perform substantially the samebiological and/or pharmacological activity in substantially the sameway. The invention is also intended to encompass biologically and/orpharmacologically active truncated versions clearly derived from thesequences disclosed and characterized herein (e.g., evidenced domainsdescribed infra) as well as chimeric sequences which contain one or moreof them.

The nucleic acid sequence also provides for the design of antisensemolecules, examples of which are provided, which are useful indownregulating, diminishing, or eliminating expression of the genomicnucleotide sequence in cells including leukocytes, endothelial cells,and tumor or cancer cells.

The human signal-transduction kinase molecule of the present inventioncan also be used in screening assays to identify antagonists orinhibitors which bind, emulate substrate, or otherwise inactivate orcompete with the biomolecule. The novel kinase can also be used inscreening assays to identify agonists which induce the production of orprolong the lifespan of the molecule in vivo or in vitro.

The invention also relates to pharmaceutical compounds and compositionscomprising the kinase molecule substantially as depicted in SEQ ID NO:3,or fragments thereof, antisense molecules capable of disruptingexpression of the naturally occurring gene, and agonists, antibodies,antagonists or inhibitors of the native signal-transduction kinase.These compositions are useful for the prevention or treatment ofconditions associated with abnormal expression of the signaltransduction molecule such as descrided infra.

pharmacological significance

Compounds which are able to modulate the activity of specific signaltransduction molecules integral to specific intracellular pathways areexpected to have significant potential for the ability to control orattenuate downstream physiological responses. Significant evidence hasbeen provided that stress kinase activation pathways are responsible forbiological effects across a wide variety of disease areas. As MAPkinases play a central role in signaling events which mediate cellularresponse to stress; compounds which modulate or inactivate specificintegral signal transduction molecules, i.e., the novel humanstress-activated serine/threonine signal transduction kinase moleculedescribed herein, SEQ ID NO:3, and nucleic acid sequences codingtherefor, e.g. SEQ ID NO:1 and SEQ ID NO:2, have significant potentialfor the ablity attenuate pathophysiological responses. Accordingly, theability to screen for antagonists and agonists which modulate theactivity of the native human stress-activated serine/threonine signaltransduction kinase molecule described herein is significantly valuabletoward the identification and development of therapeutic agents.

Potential diagnostic and therapeutic applications are readily apparentfor modulators of the human Ste20-like signal transductionserine/threonine kinase described herein. Areas which are common todisease particularly in need of therapeutic intervention include but arenot limited to pathophysiological disorders manifested by dysfunctionalleukocytes, T-cell activation, acute and chronic inflammatory disease,auto-immune disorders, rheumatoid arthritis, osteoarthritis, transplantrejection, macrophage regulation, endothelial cell regulation,angiogenesis, atherosclerosis, fibroblasts regulation, pathologicalfibrosis, asthma, allergic response, ARDS, atheroma, osteoarthritis,heart failure, cancer, diabetes, obeisity, cachexia, Alzheimers, sepsis,neurodegeneration, and related disorders.

Oxidants, mechanical stress, UV irradiation, and immunologic mediatorslead to the activation of proinflammatory gene products,apoptosis-related genes, and acute phase response genes. These responsesare the underlying causes of vascular injury and inflammation. In viewof the recent literature, it is generally accepted that MAPK pathwaysincluding the SAPK/JNK and p38 kinase pathways, for example, are centralcomponents in the response of cells to chemical, mechanical andproinflammatory assaults. With such a prominent role in cell activation,stress kinases are likely to play an important role in disease areasincluding, but not limited to, stroke (ischemia reperfusion), ARDS,sepsis, neurodegeneration, and inflammation. During reperfusion ofischemic tissue, for instance, there is a marked increase in stressactivated protein kinase activity leading to c-Jun/ATF-2 activation toenhance gene transcription leading to either apoptosis ordifferentiation and cell repair. In the inflammatory response, TNF-α andIL-1β activate p38, which triggers production of more TNF and IL-1,which amplifies the inflammatory response. This process is thought toplay a role in both septic shock and formation of the atherscleroticplaque. SAPKs are also thought to play a role in the induction ofE-selectin expression in endothelial cells and matrix metalloproteinasesin inflammatory cells, indicating a role for SAPKs in postischemicinjury and tissue remodeling. Moreover, SAPK-p46β₁, a brain-specifickinase, colocalizes with the prominent Alzheimer's disease marker,ALZ-50, suggesting that the proline-directed hyperphosphorylation of tauprotein is catalyzed by this kinase.

The signal-transduction kinase homolog described herein (SEQ ID NO:3) isbelieved to transduce cellular response to stressors leading to theactivation of proinflammatory gene products. The deleterious effects ofthe mediators of inflammation, including ROS and cytokines, open newavenues for the development of original anti-inflammatory therapies. AsMAP kinases play a central role in signaling events which mediatecellular response to stress, their inactivation or antagonization is keyto the attenuation of the response. Clear evidence has been shown, forinstance, that ERK and JNK pathways are strongly linked to IL-2production. ERK and JNK pathways are clearly established to be requiredfor full T-lymphocyte activation leading to IL-2 gene transcription andT-cell proliferation. Interruption of the signaling process by selectivekinase inhibition is therefore expected to reduce IL-2 production andT-lymphocyte proliferation which would be therapeutically beneficial inchronic inflammatory diseases. MAPKs including stress activated kinasesare expected to play key roles in the pathology of autoimmune diseasesincluding but not limited to rheumatoid arthritis (RA), osteoarthritis(OA), and multiple sclerosis (MS). Accordingly, the novel humanserine/threonine stress-activated kinase signal transduction moleculedescribed herein (SEQ ID NO:3), and nucleic acid sequences codingtherefor, e.g. SEQ ID NO:1 and SEQ ID NO:2, have significant potentialas a specific targets that can be exploited diagnostically andtherapeutically for the control of dysfunctional leukocytes, includingbut not limited to dysfunctional T-lymphocytes, and in the treatment ofautoimmune disease including rheumatoid arthritis (RA) andosteoarthritis (OA), and in the study, diagnosis, and treatment of acuteand chronic inflammation including but not limited to conditions such asasthma and ARDS as well as other diseases manifested by dysfunctionalleukocytes.

Excess production of oxidants is responsible for the activation of MAPKpathways. Excess production of oxidants is also common to the majoratherosclerotic risk factors as well as to ischaemic reperfusion injury.Lipid peroxidation in cell membranes, cytoplasmic free radicals,together with `oxidative stress` lead to activation of AP-1 and NF-KB.The JNK pathway has been established as well as activation of Rasappears to be involved in the responses. Stimulation of macrophages andendothelial cells by LPS or TNF/IL-1 results in the activation of MAPK,JNK and P38 pathways. The selective P38 inhibitor SB203580, for example,has been clearly shown to inhibit production of TNF and IL-1 byLPS-stimulated macrophages as well as TNF, IL-6 and IL-8 byTNF-stimulated HUVECs. Philip Cohen, Dissection of Protein KinaseCascades That Mediate Cellular Response to Cytokines and CellularStress, in: Intracellualr Signal Transduction, Advances in Pharmacology,Vol. 36, Academic Press (1996). MAPK pathways are also activated byshear stress. Atherosclerotic lesions develop and progress in areas oflow and unstable shear stress and not in areas exposed to steady shear.Acute changes in shear stress activate MAPK pathways and chronic stressdesensitizes MAPK and NF-kB pathways indicating that these pathways areactivated.

Transient shear stress can activate inflammatory genes partly throughactivation of the JNK pathway and AP-1 although the ERK pathway is alsoactivated. Despite major efforts to develop new therapeutic approachesAdult Respiratory Distress Syndrome (ARDS) (acute pulmonary inflammationcharacterized by the massive generation of Reactive Oxygen Species (ROS)within the lung) remains lethal for about 50% of affected patients.Polla. B. S., et al., Stress Proteins in Inflammation, in: StressInducible Cellular Responses, Feige, U., et al. Eds., Birkhauser Verlag(1996). Accordingly, the novel human serine/threonine stress-activatedkinase signal transduction molecule described herein (SEQ ID NO:3), andnucleic acid sequences coding therefor, e.g., SEQ ID NO:1 and SEQ IDNO:2, have significant potential as specific targets that can beexploited diagnostically and therapeutically for the control ofpathophysiologies relating to inflammation, dysfunctional macrophages,dysfunctional endothelial cells, and related inflammatory diseasesincluding but not limited to conditions such as atherosclerosis, asthma,allergic response, ARDS, heart failure, Atheroma and related disorders.

Differentiation, proliferation, growth arrest, or apoptosis of cellsdepends on the presence of appropriate cytokines and their receptors, aswell as the corresponding cellular signal transduction cascades. Itremains clear that stress kinase pathways, make critical contributionsto transformation. In view of the positioning of raf-MEK1-Erk1/2downstream of ras, the antiproliferative biological effect of inhibitingsignal transduction in the MAP kinase pathway have significanttherapeutic potential, applicable, for example, but not limited totumours harbouring oncogenic ras. Moreover, activation of stress pathwaykinases has been implicated in mediating apoptosis by agents such as TNFin model systems. Evidence regarding the involvement of intracellularsignalling pathways in the maintenance of cellular survival suggeststhat survival and mitogenic signals can be separable and that thebalance between these signals plays a key role in determining the fateof transformed cells. There are also indications that perturbation ofthis balance provides selective apoptosis. It would be critical toachieve optimum selectivity between pathways to achieve apoptosispromotion in tumour cells.

Unfortunately, in spite of the introduction of numerous new drugs duringthe last three decades, only very modest progress can be registered withregard to both cure or survival rates of cancer patients treated withanti-tumor agents. Thus, there is a need for new, more efficient andless toxic compounds. The majority of presently used anti-tumor agentsinterfere with the biosynthesis of nucleic acids or their intracellularfunction. Compounds which inhibit uncontrolled growth by interferingwith mitogenic signal transduction may act as cytostatic rather thancytotoxic drugs. Furthermore, attenuation of cellular proliferation hasfrequently been shown to cause tissue differentiation. Finally, blockadeof mitogenic stimulation in a cell can result in the induction ofapoptosis or programmed cell death. Accordingly, the novel humanserine/threonine stress-activated kinase signal transduction moleculedescribed herein (SEQ ID NO:3), and nucleic acid sequences codingtherefor, e.g. SEQ ID NO:1 and SEQ ID NO:2, have significant potentialas a specific targets that can be exploited diagnostically andtherapeutically to control cancer and the proliferation of tumor cells.

Activation of members of each of the MAPK pathways has been demonstratedin response to endothelin, serum, PDGF and TGFP in various types of`fibroblast`. Dominant negatives of ERK1 and Rac have been demonstrated,for example, to inhibit expression of a collagen promotor/reporter genein TGFβ-stimulated 3T3 fibroblasts. Moreover, in stellate liver cells,for instance, evidence has been shown that Raf and the JNK pathwayinteract to control cell proliferation and collagen expression. A numberof cytokines, some of which are known to activate SAPKs in cells, havebeen implicated in cachexia. Accordingly, the novel humanserine/threonine stress-activated kinase signal transduction moleculedescribed herein (SEQ ID NO:3), and nucleic acid sequences codingtherefor, e.g. SEQ ID NO:1 and SEQ ID NO:2, have significant potentialas specific targets that can be exploited diagnostically andtherapeutically in the control of dysfunctional fibroblasts, includingbut not limited to conditions such as pathological fibrosis, andcachexia as well as other diseases manifested by dysfunctionalfibroblasts.

The establishment and remodeling of blood vessels is controlled byparacrine signals, many of which are protein ligands that bind andmodulate the activity of transmembrane receptor tyrosine kinases (RTKs).The basic view of RTK signaling has come from studies (performed largelyin fibroblasts) of ligand-dependent autophosphorylation and activationof the branched Ras pathways. The results suggest that most RTKs aresimilarly coupled into the intracellular signal transduction cascade andare capable of inducing cell proliferation. Hanahan, D., SignalingVascular Morphogenesis and Maintenance, Science, 227:48 (1997).Angiogenic response of vascular endothelium, endothelial cellproliferation, is one of the first steps in the angiogenic process whichhas clearly been demonstrated to be induced by hypoxia stress. VEGF,bFGF, and EGF all have been demonstrated to upregulate MAPK in HUVECcells. Accordingly, the novel human serine/threonine stress-activatedkinase signal transduction molecule described herein (SEQ ID NO:3), andnucleic acid sequences coding therefor, e.g. SEQ ID NO:1 and SEQ IDNO:2, have significant potential as specific targets that can beexploited diagnostically and therapeutically to control angiogenesis aswell as other manifestations of dysfunctional fibroblasts.

Stress-activated protein kinase (SAPK), members of the ERK family, areactivated in situ by inflammatory stimuli, including tumour-necrosisfactor (TNF). TNF is believed to be responsible for the development ofinsulin resistance associated with obesity. Exposure of cultured cellsto TNFα induces insulin resistance which is believed to be mediated bythe Type I TNFα receptor and intracellular signalling mechanisms. Inview of the evidence that TNFα is intimately associated with theactivation of Stress Activated Protein Kinases (SAPKs), the novel humanserine/threonine stress-activated kinase signal transduction moleculedescribed herein (SEQ ID NO:3), and nucleic acid sequences codingtherefor, e.g. SEQ ID NO:1 and SEQ ID NO:2, have significant potentialas specific targets that can be exploited diagnostically andtherapeutically to control Diabetes and related disorders.

The leptin receptor belongs to the cytokine receptor superfamily ofwhich several members have been shown to feed into SAPK pathways.Intracellular signalling pathways utilised by leptin and the potentialfor regulation of the leptin receptor through "cross-talk" with othersignalling pathways is expected to lead to the design of noveltherapeutic approaches for the treatment of obesity. Accordingly, thenovel human serine/threonine stress-activated kinase signal transductionmolecule described herein (SEQ ID NO:3), and nucleic acid sequencescoding therefor, e.g. SEQ ID NO:1 and SEQ ID NO:2, have significantpotential as specific targets that can be exploited diagnostically andtherapeutically to control Obesity and related disorders.

Compounds which modulate or inactivate specific signal transductionmolecules integral to specific cytosolic pathways generally havesignificant potential for the ability to modulate or attenuatedownstream physiological responses. Accordingly, the ability to screenfor compounds which modulate the activity of the native humanstress-activated serine/threonine signal transduction kinase moleculedescribed herein is of paramount importance toward the development oftherapeutic agents.

functional constructions

The cDNA for the kinase of the present invention has been subcloned intoseveral expression vectors as examples of providing recombinant proteinuseful for drug screen assays and/or for employment in vivo or otherwiseof the physiological role of the kinase. Expression studies areperformed using either constitutive, induced, or tissue specificexpression systems in mammalian cell lines. The constructs hereindescribed are example embodiments to be used to transfect mammalian celllines, e.g., U937 and other well-known cell lines, in order to providerecombinant protein suitable for use in drug screen assays. Constructsare contemplated for over-expressing the wild-type and/or dominantnegative kinase of the present invention for characterization itsphysiological role including activation stimuli, identification ofsignal transduction pathways, identification of protein-proteininteractions, as well as validation of optimal drug candiates.

The nucleic acid sequences which encode the novel kinase can besubcloned into an expression vector in the antisense orientation toprovide a tool for producing gene knock-out studies of gene function.Down-regulation of the native signal transduction kinase moleculedescribed herein is also contemplated using antisense expression.

SEQ ID NO:6 and SEQ ID NO:7 are oligonucleotide PCR primers used toproduce full-length nucleic acid sequence amplicon pertaining to thenovel human stress activated kinase. A shuttle construct containing anucleic acid coding region of the novel signal transduction kinase wasproduced by "TA" cloning of the PCR amplicon (SEQ ID NO:8), describedinfra, into the pCR2.1 vector (Invitrogen, Carlsbad, Calif.).

A GST-Kinase fusion expression vector, designed to express a N-terminalGlutathione S-transferase fusion protein (produced using pGEX5×1,Pharmacia Biotech, Inc., Piscataway, N.J.), was constructed bysubcloning the signal transduction kinase cDNA from the pCR2.1 vectorinto the pGEX5×1 vector. The signal transduction kinase cDNA was excisedfrom the pCR2.1 vector by restriction digestion using EcoRI and EcoICRI.The fragment was gel purified, then inserted into pGEX5×1 which had beendigested with EcoRI and SmaI restriction endonucleases and gel purified.Insertion of the signal transduction kinase cDNA into pGEX5×1 yielded achimeric EcoICRI:SmaI site which was no longer recognizable by eitherrestriction enzyme. The ligated DNA was used to transform DH5α competentE. coli cells (Life Technologies, Gaithersburg, Md.). Single, isolatedtransformed E. coli colonies were grown in selective media (LB broth,carbenicillin) overnight at 37° C. and subsequently used to prepareplasmid DNA (Qiagen Plasmid DNA preparation kit). Clones were sequenced(ABI PRISM™ Dye Terminator Cycle sequencing on ABI PRISM™377 automatedsequencer) to identify correctly inserted cDNA. A correctly clonedconstruct was selected and used to transform BL21 competent E. colicells (Novagen, Inc.) for expression. Expression and purification of theGST/human kinase fusion protein is described infra in EXAMPLE IX.

kinase activity

A kinase activity assay was used to demonstrate the novel signaltransduction kinase SEQ ID NO:3 is a functional kinase. RecombinantGST-SEQ ID NO:3 was expressed in BL21 (DE3) E. coli cells and purifiedas outlined in Example IX. The recombinant kinase was tested forcatalytic activity as shown in Example II. GST in place of GST-SEQ IDNO:3 kinase shows no phosphorylation and indicates that phosphorylationin the presence of SEQ ID NO:3 is due to its kinase activity and not dueto a co-purified contaminant.

To construct a His₆ -Xpress epitope tagged novel human signaltransduction kinase mammalian expression vector the novel kinase proteincoding region was excised from the pCR2.1 construct using EcoRI (5') andEcoICRI (3') and inserted into the pcDNA3.1HisC vector (Invitrogen) atthe EcoRI (5') and EcoRV (3') sites. Joining of the EcoICRI site fromthe pCR2.1 contruct to the EcoRV site from the pcDNA3.1HisC vectorresults in the loss of both sites due to a blunt ended ligation. Theresulting construct encodes an epitope tagged kinase protein containing6 histidine residues and the "Xpress" antibody epitope at the N-terminusof the novel kinase protein. Expression of the epitope tagged novelsignal transduction kinase in eukaryotic cells is effected bytansfecting mammalian cell lines either transiently or stably withexpression vectors containing the novel kinase coding region. Thetransformed cells serve as sources of kinase for screening candidatecompound modulators, inter alia.

generally acceptable vectors

In accordance with the present invention, polynucleotide sequences whichencode the novel kinase, fragments of the polypeptide, fusion proteinsor functional equivalents thereof may be used in recombinant DNAmolecules that direct the expression of the signal transduction moleculein appropriate host cells. Due to the inherent degeneracy of the geneticcode, other DNA sequences which encode substantially the same or afunctionally equivalent amino acid sequence, may be used to clone andexpress the novel human signal-transduction kinase. As will beunderstood by those of skill in the art, it may be advantageous toproduce novel kinase-encoding nucleotide sequences possessingnon-naturally occurring codons.

Specific initiation signals may also be required for efficienttranslation of a signal-transduction kinase sequence. These signalsinclude the ATG initiation codon and adjacent sequences. In cases wherethe novel kinase, its initiation codon and upstream sequences areinserted into the appropriate expression vector, no additionaltranslational control signals may be needed. However, in cases whereonly coding sequence, or a portion thereof, is inserted, exogenoustranscriptional control signals including the ATG initiation codon mustbe provided. Furthermore, the initiation codon must be in the correctreading frame to ensure transcription of the entire insert. Exogenoustranscriptional elements and initiation codons can be of variousorigins, both natural and synthetic.

Cloned signal transduction kinase cDNA obtained through the methodsdescribed herein may be recombinantly expressed by molecular cloninginto an expression vector containing a suitable promoter and otherappropriate transcription regulatory elements, and transferred intoprokaryotic or eukaryotic host cells to produce the kinase polypeptide.Techniques for such manipulations are fully described in Sambrook, J.,et al., Molecular Cloning Second Edition, Cold Spring Harbor Press(1990), and are well known in the art.

Expression vectors are described herein as DNA sequences for thetranscription of cloned copies of genes and the translation of theirmRNAs in an appropriate host cell. Such vectors can be used to expressnucleic acid sequences in a variety of hosts such as bacteria, bluegreenalgae, plant cells, insect cells, fungal cells, human, and animal cells.Specifically designed vectors allow the shuttling of DNA between hostssuch as bacteria-yeast, or bacteria-animal cells, or bacteria-fungalcells, or bacteria-invertebrate cells.

A variety of mammalian expression vectors may be used to express therecombinant human kinase molecule disclosed herein in mammalian cells.Commercially available mammalian expression vectors which are suitablefor recombinant expression, include but are not limited to, pcDNA3(Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5(Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2) (ATCC 37110),pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and IZD35 (ATCC37565), pLXIN and pSIR (CLONTECH), pIRES-EGFP (CLONTECH). INVITROGENcorporation provides a wide variety of commercially available mammalianexpression vector/systems which can be effectively used with the presentinvention. INVITROGEN, Carlsbad, Calif. See, also, PHARMINGEN products,vectors and systems, San Diego, Calif.

Baculoviral expression systems may also be used with the presentinvention to produce high yields of biologically active protein. Vectorssuch as the CLONETECH, BacPak™ Baculovirus expression system andprotocols are preferred which are commercially available. CLONTECH, PaloAlto, Calif. Miller, L. K., et al., Curr. Op. Genet. Dev. 3:97 (1993);O'Reilly, D. R., et al., Baculovirus Expression Vectors: A LaboratoryManual, 127. Vectors such as the INVITROGEN, MaxBac™ Baculovirusexpression system, insect cells, and protocols are also preferred whichare commercially available. INVITROGEN, Carlsbad, Calif.

example host cells

Host cells transformed with a nucleotide sequence which encodes thehuman kinase of the present invention may be cultured under conditionssuitable for the expression and recovery of the encoded protein fromcell culture. Particularly preferred embodiments of the presentinvention are host cells transformed with a purified polynucleotidecomprising a nucleic acid sequence encoding the polypeptide having thesequence substantially as depicted in SEQ ID NO:3 or a biologicallyactive fragment thereof. Cells of this type or preparations made fromthem may be used to screen for pharmacologically active modulators ofthe novel human signal-transduction kinase activity.

Eukaryotic recombinant host cells are especially preferred. Examplesinclude but are not limited to yeast, mammalian cells including but notlimited to cell lines of human, bovine, porcine, monkey and rodentorigin, and insect cells including but not limited to Drosophila andsilkworm derived cell lines. Cell lines derived from mammalian specieswhich may be suitable and which are commercially available, include butare not limited to, L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCCCCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70),COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3(ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C1271 (ATCCCRL 1616),BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).

The expression vector may be introduced into host cells expressing thenovel kinase via any one of a number of techniques including but notlimited to transformation, transfection, lipofection, protoplast fusion,and electroporation. Commercially available kits applicable for use withthe present invention for hererologous expression, includingwell-characterized vectors, transfection reagents and conditions, andcell culture materials are well-established and readily available.CLONTECH, Palo Alto, Calif.; INVITROGEN, Carlsbad, Calif.; PHARMINGEN,San Diego, Calif.; STRATAGENE, LaJolla, Calif. The expressionvector-containing cells are clonally propagated and individuallyanalyzed to determine the level of novel kinase protein production.Identification of host cell clones which express the novel kinase may beperformed by several means, including but not limited to immunologicalreactivity with antibodies described herein, and/or the presence of hostcell-associated specific kinase activity, and/or the ability tocovalently cross-link specific substrate to the novel kinase with thebifunctional cross-linking reagent disuccinimidyl suberate or similarcross-linking reagents.

The signal transduction molecule of the present invention may also beexpressed as a recombinant protein with one or more additionalpolypeptide domains added to facilitate protein purification. Suchpurification facilitating domains include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals (Porath, J., Protein Exp. Purif.3:263 (1992)), protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle Wash.). The inclusion of acleavable linker sequences such as Factor XA or enterokinase(Invitrogen, San Diego Calif.) between the purification domain and TMPis useful to facilitate purification.

Systems such as the CLONTECH, TALON™ nondenaturing protein purificationkit for purifying 6xHis-tagged proteins under native conditions andprotocols are preferred which are commercially available. CLONTECH, PaloAlto, Calif.

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing which cleaves a nascent form of theprotein may also be important for correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, 293, WI38,NIH-3T3, HEK293 etc., have specific cellular machinery andcharacteristic mechanisms for such post-translational activities and maybe chosen to ensure the correct modification and processing of theintroduced, foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe novel kinase may be transformed using expression vectors whichcontain viral origins of replication or endogenous expression elementsand a selectable marker gene. Following the introduction of the vector,cells may be allowed to grow for 1-2 days in an enriched media beforethey are switched to selective media. The purpose of the selectablemarker is to confer resistance to selection, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clumps of stably transformed cells can beproliferated using tissue culture techniques appropriate to the celltype.

The human kinase can be produced in the yeast S. cerevisiae followingthe insertion of the optimal cDNA cistron into expression vectorsdesigned to direct the intracellular or extracellular expression of theheterologous protein. In the case of intracellular expression, vectorssuch as EmBLyex4 or the like are ligated to the beta subunit cistron.See, e.g., Rinas, U., et al., Biotechnology, 8:543 (1990); Horowitz, B.,et al., J. Biol. Chem., 265:4189 (1989). For extracellular expression,the kinase cistron is ligated into yeast expression vectors which mayemploy any of a series of well-characterized secretion signals. Thelevels of expressed novel kinase are determined by the assays describedherein.

A variety of protocols for detecting and measuring the expression of thenovel kinase, using either polyclonal or monoclonal antibodies specificfor the protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopesmay be employed. Well known competitive binding techniques may also beemployed. See, e.g., Hampton, R., et al. (1990), Serological Methods--aLaboratory Manual, APS Press, St Paul Minn.; Maddox, D. E., et al., J.Exp. Med. 158:1211.

screening assays

The present invention is also directed to methods for screening forcompounds which modulate the expression of DNA or RNA encoding the novelkinase polypeptide, as well as the function of the signal-transductionkinase polypeptide in vivo. Compounds that modulate the expression ofDNA or RNA encoding the signal-transduction kinase polypeptide or thefunction of the polypeptide may be detected by a variety of assays. Theassay may be a simple "yes/no" assay to determine whether there is achange in expression or function. The assay may be made quantitative bycomparing the expression or function of a test sample with the levels ofexpression or function in a standard sample.

The signal-transduction kinase described herein, its immunogenicfragments or oligopeptides can be used for screening therapeuticcompounds in any of a variety of drug screening techniques. The fragmentemployed in such a test may be free in solution, affixed to a solidsupport, borne on a cell surface, or located intracellularly. Theabolition of activity or the formation of binding complexes, between thesignal-transduction kinase polypeptide and the agent being tested, maybe measured. Accordingly, the present invention provides a method forscreening a plurality of compounds for specific binding affinity withthe signal-transduction kinase polypeptide or a fragment thereof,comprising providing a plurality of compounds; combining thesignal-transduction kinase polypeptide of the present invention or afragment thereof with each of a plurality of compounds for a timesufficient to allow binding under suitable conditions; and detectingbinding of the kinase polypeptide, or fragment thereof, to each of theplurality of compounds, thereby identifying the compounds whichspecifically bind the signal-transduction kinase polypeptide.

Methods of identifying compounds that modulate the activity of asignal-transduction kinase polypeptide are generally preferred, whichcomprise combining a candidate compound modulator of asignal-transduction kinase activity with a polypeptide of asignal-transduction kinase having the sequence substantially as depictedin SEQ ID NO:3, and measuring an effect of the candidate compoundmodulator on the kinase activity.

Polypeptides which comprise the human signal-transduction kinase, SEQ IDNO:3, its functional fragments or oligopeptides including but notlimited to SEQ ID NO:3 positions 24-274, and SEQ ID NO:3 positions275-416, as well as variants contemplated herein can be used forscreening prospective therapeutic compounds in any of a variety of drugscreening techniques.

Methods of identifying compounds that modulate a biological and/orpharmacological activity of a signal transduction kinase polypeptide aregenerally preferred which comprise combining a candidate compoundmodulator with a purified polypeptide comprising the amino acid sequenceas depicted in SEQ ID NO:3 or a variant of SEQ ID NO:3 having at leastabout 90% homology to a member selected from the group consistingessentially of: (SEQ ID NO:3, SEQ ID NO:3 positions 24-274, and SEQ IDNO:3 positions 275-416) and measuring an effect of the candidatecompound modulator on the biological and/or pharmacological activity ofthe polypeptide. See, Examples.

A further method of identifying compounds that modulate a biologicaland/or pharmacological activity of a polypeptide comprising the aminoacid sequence as depicted in SEQ ID NO:3 or a variant of SEQ ID NO:3having at least about 90% homology to a member selected from the groupconsisting essentially of: (SEQ ID NO:3, SEQ ID NO:3 positions 24-274,and SEQ ID NO:3 positions 275-416) comprises combining a candidatecompound modulator with a host-cell which expresses the signaltransduction kinase polypeptide, and measuring an effect of thecandidate compound modulator on the biological and/or pharmacologicalactivity of the polypeptide.

A filter assay based on the protocol of Reuter et al. (1995) is alsoused to screen for compounds which modulate the activity of the novelkinase described herein: Starting with MBP coated 96-well FlashPlates®(NEN™ Life Science Products) reaction buffer (3X kinase reaction buffer(KRB) contains: 60 mM HEPES (pH 7.5), 30 mM magnesium acetate, 0.15 mMATP, 3 mM DTT, 0.03 mM sodium orthovanadate) is added, 0.25 μCi [γ³³P]-ATP at a concentration no greater than 1 μg/ml, (determined bytitration of individual enzyme preparations for a concentration thatallows kinetic determinations over a 1 hour time course of the humankinase) of the human kinase are added to each well and incubated 1 hourat 30° C. in the presence or absence of 10 μM test compound. Totalreaction volume is 100 μL. The reaction is stopped by the addition ofEDTA (pH 7.0) to a final concentration of 80 mM. The samples arecentrifuged and 50 μL of the supernatant spotted on p81 cation-exchangefilter paper (Whatman, No. 3698 915). The filters are then washed 3times in 200 mL of 180 mM H₃ PO₄ (5-10 min each), and once in 200 mL of96% ethanol. After air drying the filters, radioactivity is determinedby Cerenkov counting in a scintillation counter. Compounds which inhibitkinase activity ≧50 percent at 10 μM are indicated by a >50% reductionin scintillation counts. Specificity and selectivity studies isdetermined by titration of inhibitory compounds to determine the IC₅₀(or other standard quantitation well known in the art for comparison)and by the substitution of other kinases in the assay. For example,determination of relative inhibitory activity of the kinase incomparison to recombinant SOK-1 and/or mst-3, expressed and isolated ina similar manner, assayed under similar conditions, provides selectivitydata. Reuter, C. W. M., Catling, A. D. and Weber, M. J., Immune ComplexKinase Assays for Mitogen-Activated Protein Kinase and MEK, Methods InEnzymology, 255:245 (1995).

See EXAMPLES VI and VII.

To evaluate the ability of a candidate agent to inhibit human tumorgrowth, human tumor cells are injected into SCID mice (severe combinedimmunodeficiency) to form palpable tumor masses. The effects of ancandidate agent in inhibiting tumor growth can be determined as follows.Approximately 1×10⁷ cells of the CCL 221 cell line (ATCC, Rockville,Md.), a human ras-dependent colon adenocarcinoma cell line, is suspendedin 100 μl DMEM and injected subcutaneously into SCID mice, such that twotumors per mouse are formed. SCID mice receive CCL 221 cells and thetumors are grown for 7 days without treatment; on the 7th day (Day 0)tumor maximal diameters and animal weights are recorded and the meantumor size for the mice is determined. On Day 1 (eight days followingtumor cell injection), treatment of the mice with candidate agent orvehicle alone is begun. One group of the mice (controls) are injectedintraperitoneally with 0.2 ml of vehicle and a second group of micereceived agent by intraperitoneal injection. Various doses of agent canbe tested in separate groups of mice. On Day 7 and Day 14, animal weightand maximal tumor diameter is measured. Average maximal tumor size foreach group on Day 0, Day 7, and Day 14 are compared Day 14, one highdose animal was followed for an additional to determine whether theagent produces a dose-dependent inhibition of tumor growth. Toxicityeffects can be examined by tracking mice weight and by harvesting lungs,livers, and spleens of the animals for histological staining.

Compounds which are identified generally according to methods descibed,contemplated, and referenced herein that modulate the biological and/orpharmacological activity of a signal-transduction molecule of thesequence substantially as depicted in SEQ ID NO:3 are especiallypreferred embodiments of the present invention.

A method of modulating a biological and/or pharmacological activity of asignal transduction kinase in a cell, tissue, or organism is preferredwhich comprises administering an effective amount of a polynucleotidecontemplated herein. `Polynucleotide` includes a polynucleotidecomprising a nucleic acid sequence which encodes a polypeptidecomprising an amino acid sequence having at least about 90% homology toSEQ ID NO:3, SEQ ID NO:3 positions 24-274, and SEQ ID NO:3 positions275-416; as well as a polynucleotide comprising a nucleic acid sequencewhich encodes a polypeptide comprising an amino acid sequence whereinone or more positions corresponding to SEQ ID NO:3, selected from thegroup consisting of (position 31 (glycine), 33 (glycine), 36 (glycine),38 (valine), 51 (alanine), 53 (lysine), 144 (aspartic acid), 149(asparagine), 162 (aspartic acid), 163 (phenylalanine), 164 (glycine),182 (threonine), 189 (glutamic acid), and 201 (aspartic acid)), aresubstituted or deleted; as well as antisense molecules which arecomplementary to a region within SEQ ID NO:1 positions 157-232 or1405-1480, example therapeutic embodiments of which are set forth supra.

An especially preferred embodiment of the present invention is a methodfor treatment of a patient in need of such treatment for a conditionwhich is mediated by the human signal-transduction kinase describedherein comprising administration of a therapeutically effective amountof a human signal-transduction kinase modulating compound.

yeast 2-hybrid system

In another embodiment of the invention, a nucleic acid sequence whichencodes a human signal-transduction kinase molecule substantially asdepicted in SEQ ID NO:3 or a biologically active fragment thereof may beligated to a heterologous sequence to encode a fusion protein. Forexample, for screening compounds for modulation of biological activity,it may be useful to encode a chimeric kinase molecule as describedherein for expression in hererologous host cells. Chimeric constructsmay also be used to express a `bait`, according to methods well knownusing a yeast two-hybrid system, to identify accessory native peptidesthat may be associated with the novel human signal-transduction kinasemolecule described herein. Fields, S., et al., Trends Genet., 10:286(1994); Allen, J. B., et al., TIBS, 20:511 (1995). A yeast two-hybridsystem has been described wherein protein:protein interactions can bedetected using a yeast-based genetic assay via reconstitution oftranscriptional activators. Fields, S., Song, O., Nature 340:245 (1989).The two-hybrid system used the ability of a pair of interacting proteinsto bring a transcription activation domain into close proximity with aDNA-binding site that regulates the expression of an adjacent reportergene. Commercially available systems such as the CLONTECH, Matchmaker™systems and protocols may be used with the present invention. CLONTECH,Palo Alto, Calif. See also, Mendelsohn, A. R., Brent, R., Curr. Op.Biotech., 5:482 (1994); Phizicky. E. M., Fields, S., MicrobiologicalRev., 59(1):94 (1995); Yang, M., et al., Nucleic Acids Res., 23(7):1 152(1995); Fields, S., Sternglanz, R., TIG, 10(8):286 (1994); and U.S. Pat.No. 5,283,173, System to Detect Protein-Protein Interactions, and U.S.Pat. No. 5,468,614, which are incorporated herein by reference.

purification via affinity columns

It is readily apparent to those skilled in the art that methods forproducing antibodies may be utilized to produce antibodies specific forthe human kinase polypeptide fragments, or the full-length nascent humankinase polypeptide. Specifically, it is readily apparent to thoseskilled in the art that antibodies may be generated which are specificfor the fully functional receptor or fragments thereof.

Kinase polypeptide antibody affinity columns are made by adding theantibodies to Affigel-10 (Biorad), a gel support which is activated withN hydroxysuccinimide esters such that the antibodies form covalentlinkages with the agarose gel bead support. The antibodies are thencoupled to the gel via amide bonds with the spacer arm. The remainingactivated esters are then quenched with 1M ethanolamine HCl (pH 8). Thecolumn is washed with water followed by 0.23M glycine HCl (pH 2.6) toremove any non-conjugated antibody or extraneous protein. The column isthen equilibrated in phosphate buffered saline (pH 7.3) with appropriatedetergent and the cell culture supernatants or cell extracts containinghuman signal-transduction kinase polypeptide made using appropriatemembrane solubilizing detergents are slowly passed through the column.The column is then washed with phosphate buffered saline/detergent untilthe optical density falls to background, then the protein is eluted with0.23M glycine-HCl (pH 2.6)/detergent. The purified humansignal-transduction kinase polypeptide is then dialyzed againstphosphate buffered saline/detergent.

Recombinant kinase molecules can be separated from other cellularproteins by use of an immunoaffinity column made with monoclonal orpolyclonal antibodies specific for full length nascent human kinasepolypeptide, or polypeptide fragments of the kinase molecule.

Human kinase polypeptides described herein may be used to affinitypurify biological effectors from native biological materials, e.g.disease tissue. Affinity chromatography techniques are well known tothose skilled in the art. A human signal-transduction kinase peptidedescribed herein or an effective fragment thereof, is fixed to a solidmatrix, e.g. CNBr activated Sepharose according to the protocol of thesupplier (Pharmacia, Piscataway, N.J.), and a homogenized/bufferedcellular solution containing a potential molecule of interest is passedthrough the column. After washing, the column retains only thebiological effector which is subsequently eluted, e.g., using 0.5Macetic acid or a NaCl gradient.

antisense molecules

Various nucleic acid sequences complementary to SEQ ID NO:1 and/or SEQID NO:2 provided herein may be used in another embodiment of theinvention to modulate the expression of a serine threonine kinase or abiological function of a downstream signal transduction molecule ortranscriptional activator, by affecting the transcription and/ortranslation of sequences corresponding to SEQ ID NO:1 and/or SEQ ID NO:2in cells. Pharmacological activity of the endogenous gene may bemodulated by affecting the transcription and/or translation, forexample, of the endogenous gene by use or administration of anti-senseconstructs to produce anti-sense transcripts or by direct delivery ofanti-sense oligomers. Antisense constructs and oligomers may each beused as embodiments of the present invention and each are related totherapeutic method embodiments practiced via direct administration asdefined herein. Example species are provided.

Antisense molecules which comprise oligomers in the range from about 12to about 25 nucleotides which are complementary to regions of SEQ IDNO:1 and/or SEQ ID NO:2 are preferred embodiments of the invention.Antisense molecules comprising oligomers from about 12 to about 25nucleotides in length which are complementary to a region within the SEQID NO:1 positions 157-232 or 1405-1480 are particularly preferredembodiments. Oligonucleotides which comprise sequences complementary tothe following positions of SEQ ID NO:1 are therefore example embodimentsof the invention:

SEQ ID NO:1 positions 157-232 in progressive increments of twelve(twelve-mers), illustrated as follows: positions 157-168, 158-169,159-170, 160-171, . . . et seq (all inclusive) . . . 219-230, 220-231,221-232. This demonstrates 65 example embodiments.

SEQ ID NO:1 positions 1405-1480 in progressive increments of twelve(twelve-mers), illustrated as follows: positions 1405-1416, 1406-1417,1407-1418, 1408-1419, . . . et seq (all inclusive) . . . 1467-1478,1468-1479, 1469-1480. This demonstrates another 65 example embodiments.

Oligonucleotides which comprise sequences complementary to andhybridizable to each of the recited areas of the human serine threoninekinase mRNA are contemplated for therapeutic use. Moreover, U.S. Pat.No. 5,639,595, Identification of Novel Drugs and Reagents, issued Jun.17, 1997, wherein methods of identifying oligonucleotide sequences thatdisplay in vivo activity are thoroughly described, is hereinincorporated by reference.

Nucleotide sequences that are complementary to the serine threoninekinase encoding nucleic acid sequence can be synthesized for antisensetherapy. These antisense molecules may be DNA, stable derivatives of DNAsuch as phosphorothioates or methylphosphonates, RNA, stable derivativesof RNA such as 2'-O-alkylRNA, or other oligonucleotide mimetics. U.S.Pat. No. 5,652,355, Hybrid Oligonucleotide Phosphorothioates, issuedJul. 29, 1997, and U.S. Pat. No. 5,652,356, Inverted Chimeric and HybridOligonucleotides, issued Jul. 29, 1997, which describe the synthesis andeffect of physiologically-stable antisense molecules, are incorporatedby reference. Serine threonine kinase antisense molecules may beintroduced into cells by microinjection, liposome encapsulation or byexpression from vectors harboring the antisense sequence. Antisensetherapy may be particularly useful for the treatment of diseases whereit is beneficial to modulate the effective biological and/orpharmacological activity of the serine threonine kinase presentedherein.

gene therapy

A human signal-transduction kinase polypeptide described herein mayadministered to a subject via gene therapy. Moreover, a polypeptide ofthe present invention may be delivered to the cells of target organs inthis manner. Conversely, signal-transduction kinase polypeptideantisense gene therapy may be used to reduce the expression of thepolypeptide in the cells of target organs. The human signal-transductionkinase polypeptide coding region can be ligated into viral vectors whichmediate transfer of the kinase polypeptide DNA by infection of recipienthost cells. Suitable viral vectors include retrovirus, adenovirus,adeno-associated virus, herpes virus, vaccinia virus, polio virus andthe like. See, e.g., U.S. Pat. No. 5,624,820, Episomal Expression Vectorfor Human Gene Therapy, issued Apr. 29, 1997. Nucleic acid codingregions of the present invention are incorporated into effectiveeukaryotic expression vectors, which are directly administered orintroduced into somatic cells for gene therapy (a nucleic acid fragmentcomprising a coding region, preferably mRNA transcripts, may also beadministered directly or introduced into somatic cells). See, e.g., U.S.Pat. No. 5,589,466, issued Dec. 31, 1996. Such nucleic acids and vectorsmay remain episomal or may be incorporated into the host chromosomal DNAas a provirus or portion thereof that includes the gene fusion andappropriate eukaryotic transcription and translation signals, i.e, aneffectively positioned RNA polymerase promoter 5' to the transcriptionalstart site and ATG translation initiation codon of the gene fusion aswell as termination codon(s) and transcript polyadenylation signalseffectively positioned 3' to the coding region. Alternatively, the humansignal-transduction kinase polypeptide DNA can be transferred into cellsfor gene therapy by non-viral techniques including receptor-mediatedtargeted DNA transfer using ligand-DNA conjugates oradenovirus-ligand-DNA conjugates, lipofection membrane fusion or directmicroinjection. These procedures and variations thereof are suitable forex vivo, as well as in vivo human signal-transduction kinase polypeptidegene therapy according to established methods in this art.

compositions

Pharmaceutically useful compositions comprising the novel human kinasepolypeptide DNA, human kinase polypeptide RNA, antisense sequences, orthe human kinase polypeptide, or variants and analogs which have thehuman kinase activity or otherwise modulate its activity, may beformulated according to known methods such as by the admixture of apharmaceutically acceptable carrier. Examples of such carriers andmethods of formulation may be found in Remington's PharmaceuticalSciences (Maack Publishing Co, Easton, Pa.). To form a pharmaceuticallyacceptable composition suitable for effective administration, suchcompositions will contain an effective amount of the protein, DNA, RNA,or modulator.

Therapeutic or diagnostic compositions of the invention are administeredto an individual in amounts sufficient to treat or diagnose humansignal-transduction kinase polypeptide related disorders. The effectiveamount may vary according to a variety of factors such as theindividual's condition, weight, sex and age. Other factors include themode of administration.

The term "chemical derivative" describes a molecule that containsadditional chemical moieties which are not normally a part of the basemolecule. Such moieties may improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties mayattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart. The therapeutically effective dose can be estimated initiallyeither in cell culture assays, eg, of neoplastic cells, or in animalmodels, usually mice, rabbits, dogs, or pigs. The animal model is alsoused to achieve a desirable concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans. A therapeuticallyeffective dose refers to that amount of protein or its antibodies,antagonists, or inhibitors which ameliorate the symptoms or condition.The exact dosage is chosen by the individual physician in view of thepatient to be treated.

Compounds identified according to the methods disclosed herein may beused alone at appropriate dosages defined by routine testing in order toobtain optimal modulation of a signal-transduction kinase, or itsactivity while minimizing any potential toxicity. In addition,co-administration or sequential administration of other agents may bedesirable.

The pharmaceutical compositions may be provided to the individual by avariety of routes such as subcutaneous, topical, oral and intramuscular.Administration of pharmaceutical compositions is accomplished orally orparenterally. Methods of parenteral delivery include topical,intra-arterial (directly to the tissue), intramuscular, subcutaneous,intramedullary, intrathecal, intraventricular, intravenous,intraperitoneal, or intranasal administration. The present inventionalso has the objective of providing suitable topical, oral, systemic andparenteral pharmaceutical formulations for use in the novel methods oftreatment of the present invention. The compositions containingcompounds identified according to this invention as the activeingredient for use in the modulation of signal-transduction kinase canbe administered in a wide variety of therapeutic dosage forms inconventional vehicles for administration. For example, the compounds canbe administered in such oral dosage forms as tablets, capsules (eachincluding timed release and sustained release formulations), pills,powders, granules, elixirs, tinctures, solutions, suspensions, syrupsand emulsions, or by injection. Likewise, they may also be administeredin intravenous (both bolus and infusion), intraperitoneal, subcutaneous,topical with or without occlusion, or intramuscular form, all usingforms well known to those of ordinary skill in the pharmaceutical arts.An effective but non-toxic amount of the compound desired can beemployed as a signal-transduction kinase modulating agent.

The daily dosage of the products may be varied over a wide range from0.01 to 1,000 mg per adult human/per day. For oral administration, thecompositions are preferably provided in the form of scored or unscoredtablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0,25.0, and 50.0 milligrams of the active ingredient for the symptomaticadjustment of the dosage to the patient to be treated. An effectiveamount of the drug is ordinarily supplied at a dosage level of fromabout 0.0001 mg/kg to about 100 mg/kg of body weight per day. The rangeis more particularly from about 0.001 mg/kg to 10 mg/kg of body weightper day. Even more particularly, the range varies from about 0.05 toabout 1 mg/kg. Of course the dosage level will vary depending upon thepotency of the particular compound. Certain compounds will be morepotent than others. In addition, the dosage level will vary dependingupon the bioavailability of the compound. The more bioavailable andpotent the compound, the less compound will need to be administeredthrough any delivery route, including but not limited to oral delivery.The dosages of the human signal-transduction kinase modulators areadjusted when combined to achieve desired effects. On the other hand,dosages of these various agents may be independently optimized andcombined to achieve a synergistic result wherein the pathology isreduced more than it would be if either agent were used alone. Thoseskilled in the art will employ different formulations for nucleotidesthan for proteins or their inhibitors. Similarly, delivery ofpolynucleotides or polypeptides will be specific to particular cells andconditions.

EXAMPLES Example I

sequence construction

The novel signal transduction kinase was identified after the assemblyof EST sequences from a proprietary database and the public MerckWashington University EST sequence database. The initial identificationof these ESTs was performed by basic local alignment search tool (BLAST)analysis of the databases using the kinase subdomain VIB sequenceHRDLKPENILLD. Sequencing of two Merck Wash. U. ESTs (zb05e11, zp83b04)provided overlapping sequencing comprised of 5'UTR, open reading frame,and 3'UTR as shown in SEQ ID NO:1. Oligonucleotide primers, sense5'-GCCTCCATGGCCCACTCGCCGGTG-3' (SEQ ID NO:6) and antisense5'-GGCACATGGAGCTCATGGGTTAAGC-3' (SEQ ID NO:7), were designed based onthe sequence obtained from the Merck Wash U ESTs. The primers weredesigned to amplify a 1353 bp fragment encompassing 6 bp upstream of theATG initiation codon to 96 bp downstream of the TAA stop codon (SEQ IDNO:8). The described fragment was amplified using cDNA's derived frommRNA isolated from human lung, brain, and kidney tissue (Clontech, PaloAlto, Calif.), and from U937, KU812, and Jurkat cell lines usingAdvantage polymerase (Clontech). The PCR conditions used were: 94° C.for 1 minute followed by 20 cycles of 94° C. for 50 seconds, 65° C. for50 seconds, 72° C. for 2 minutes. The PCR amplicons were gel purifiedusing QiaexII agarose gel purification kit (Qiagen) and subcloned intothe pCR2.1 Topo "TA" vector (Invitrogen) by the "TA" cloning approach asper the kit protocols. The topoisomerase reactions were used totransform DH5a E. coli competent cells (Life Technologies). Single,isolated transformed E. coli colonies were grown in selective media (LBbroth, carbenicillin) overnight at 37° C. and subsequently used toprepare plasmid DNA (Qiagen Plasmid DNA preparation kit). Clones derivedfrom lung, brain, and kidney cDNA's were sequenced (ABI PRISM™ DyeTerminator Cycle sequencing on ABI PRISM™377 automated sequencer). Thisyeilded single cDNA species of the novel kinase which validated theexistence of this cDNA. A clone derived from lung cDNA was found to havesequence identical to that determined from the Merck Wash. U. ESTclones. The 1353 bp sequence (SEQ ID NO:8) was found to contain a 1248bp (SEQ ID NO:2) open reading frame (ORF) with a Kozak consensussequence at the initiation ATG codon. Translation of the ORF resulted ina 416 amino acid protein sequence (SEQ ID NO:3) which contains all 12conserved domains found in eukaryotic serine/threonine protein kinases.The protein has a predicted molecular weight of 46527.83 daltons, anisoelectric point of 5.097, and a net charge of -13 at pH 7.0.

Example II

assay for human kinase activity generally

Recombinant, purified GST/kinase (10 μL) is added to 20 μg myelin basicprotein (MBP) in 10 μL of a 3X kinase reaction buffer (KRB) containing:60 mM HEPES (pH 7.5), 30 mM magnesium acetate, 0.15 mM ATP, 3 mM DTT,0.03 mM sodium orthovanadate. The reaction is started by the addition of5 μCi [γ³² P] ATP (10 μL). Samples are incubated for 5 minutes at 30° C.and the reaction is stopped by addition of 4X Laemmli sample buffer.Proteins are separated on 12% Tris/glycine SDS gels, stained withCoomassie blue, dried and exposed to autoradiograph film.

specific assay

3x Kinase Reaction Buffer (KRB):

60 mM HEPES, pH 7.5

30 mM Magnesium Acetate

150 AM ATP

3 mM DTT

0.3 mM Na₃ VO₄

0.5 μg/ml Myelin Basic Protein

Assay:

1. Add 10 μL 3X KRB and 10 μL recombinant kinase (lug) to each tube.

2. Start reaction by adding 10 μL [γ-³² P] ATP (0.5 mCi/ml).

3. Incubate 30° C. for 30 minutes.

4. Stop reaction with 7.5 μL 5X Sample Buffer.

5. Boil the samples for 5 minutes then place on ice for 1 minute.

6. Load 25 μL of sample (66%) on a 10-20% Tris/glycine PAGE gel and runat 35 mA/gel for 47 minutes in Tris/glycine/SDS PAGE running buffer.

7. Stain the gel with Coomassie blue for 30-45 minutes then destainovernight in 10% methanol/7% acetic acid.

8. Fix the gel 1 hour in 3:1:6 MeOH/glacial acetic acid/H₂ O to reducebackground radiation.

9. Photograph the gel to visualize the protein bands.

10. Dry the gel and expose to film.

Example III

production of anti-kinase polyclonal antibodies

Antigenic peptide fragments were identified within the N-terminal,c-terminal and central regions of the novel human kinase utilizing awell established algorithm method developed by Jameson and Wolf. TheAntigenic Index: A Novel Algorithm for Predicting AntigenicDeterminants, CABIOS, 4:181 (1988). The algorithm carries out six majorsubroutines with the following hierarchy:

1) determination of hydrophilicity, Hopp-Woods (1981)

2) calculation of surface probability, Emini (1985)

3) prediction of backbone or chain flexibility, Karplus-Schultz (1985)

4) prediction of secondary structure, Chou-Fasman (1978)

5) prediction of secondary structure, Garnier-Robson (1978)

6) flexibility parameters and hydropathy/solvent accessibility factorsare combined to determine the antigenic index

The antigenic index was plotted for the entire molecule.

A peptide sequence, LKQQDENNASRNQA, corresponding to SEQ ID NO:3 aminoacid residue positions 364-377 was selected for synthesis and antibodyproduction based on antigenicity and uniqueness of sequence compared toother identified kinase family members. Genosys Biotechnologies, Inc.was contracted to synthesize the peptide and produce the polyclonalantisera.

Chou, P. Y. and Fasman, G. D., (1978) Prediction of the secondarystructure of proteins from their amino acid sequence, Adv. Enzymol,47:45-148; Emini, E. A., Hughes, J., Perlow, D. and Boger, J., (1985)Induction of Hepatitis A Virus-Neutralizing Antibody by a Virus-SpecificSynthetic Peptide, J. Virology, 55:836-839; Garnier, J., Osguthorpe, D.J., and Robson, B., (1978) Analysis of the accuracy and implications ofsimple method for predicting the secondary structure of globularproteins, J. Mol. Biol., 120:97-120; Harlow, E. and Lane, D., (1988)Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.; Hopp, T. P. and Woods, K. R., (1981)Prediction of Protein Antigenic Determinants from Amino Acid Sequences,Proc. Natl. Acad. Sci., 78:3824-3828; Jameson, B. A., and Wolf, H.,(1988) The antigenic index: a novel algorithm for predicting antigenicdeterminants, CABIOS 4:181-186; Karplus, P. A. and Schultz, G. E.,(1985) Prediction of chain flexibility in proteins, Naturwissenschaften,72:212-213.

Example IV

immunoprecipitation

Immunoprecipitation of the human kinase molecule described herein isperformed substantially according to the method described by Suchard, S.J., et al. J. Immunol., 158:4961 (1997). Cell lysates are combined with1 μg of either anti-enterokinase protease cleavage site/Xpress™ antibody(Invitrogen Corp.) for the recombinant kinase described herein orpeptide-specific polyclonal antibody against the native kinase describedherein. Rabbit IgG is used as a control. Samples are incubated at 4°C.≧2 hours with rotation. Immunocomplexes are incubated with protein ASepharose (Pharmacia) for 2 hours at 4° C. with rotation. The beads arewashed in buffer containing 50 mM Tris (pH 8.0), 100 mM NaCl, 1 mM Na₃VO₄, 1% Triton X-100, and Complete™ Protease Inhibitor Cocktail.Adsorbed proteins are solubilized in sample buffer and separated on 12%SDS-PAGE minigels.

Example V

northern blot analysis

A 289 bp PCR amplicon derived from the 3' end of the open reading frameand 3'UTR region of the novel cDNA (SEQ ID NO:1 base positions1240-1528) was obtained using the primers: sense5'-GTCTATGATAATCACACCTGC-3' (SEQ ID NO:9), and antisense5'-GGCACATGGAGCTCATGGGTTAAGC-3' (SEQ ID NO:10). This DNA fragment(probe) was labeled with 50 μCi α-³² P-dCTP using the Ready-to-Go™ DNALabelling Kit (Pharmacia Biotech). The labeled probe was used tohybridize against human tissue mRNA's immobilized on a solid support(Clontech Human Cancer Cell Line Multiple Tissue Northern Blot as wellas a Endothelial and Epithelial Primary Cells and Cell Lines NorthernBlot). Hybridization was performed essentially as described by Clontech(Protocol #PT-1200-1) using ExpressHyb™ solution. The Northern blotanalysis identified a primary transcript pertaining to the novel signaltransduction kinase of approximately 3.5 kb in length. Prominenttranscripts are apparent primarily in immune tissues (lymph node,peripheral blood leukocytes, spleen, fetal liver, bone marrow, thymus,and placenta).

Example VI

high throughput screening for compounds which modulate activity

High throughput screening for modulator compounds is performed using MBPcoated 96-well FlashPlates® (NEN™ Life Science Products). Kinasereaction buffer (3X kinase reaction buffer (KRB) contains: 60 mM HEPES(pH 7.5), 30 mM magnesium acetate, 0.15 mM ATP, 3 mM DTT, 0.03 mM sodiumorthovanadate) 0.25 μCi [γ³³ P]-ATP at a concentration no greater than 1μg/ml, (determined by titration of individual enzyme preparations for aconcentration that allows kinetic determinations over a 1 hour timecourse of the human kinase) are added to each well and incubated 1 hourat 30° C. in the presence or absence of 10 μM test compound. Totalreaction volume is 100 μL. Following incubation, the reaction mixture isaspirated and the wells rinsed 2 times with 300 μL PBS. Incorporation ofraiolabeled phosphate is determined by scintillation counting, PackardInstrument Co. TopCount, 12-detector, 96-well microplate scintillationcounter and luminescence counter, model B991200. Compounds which inhibitkinase activity >50 percent at 10 μM are are indicated by a >50%reduction in scintillation counts. Specificity and selectivity studiesis determined by titration of inhibitory compounds to determine the IC₅₀(or other standard quantitation well known in the art for comparison)and by the substitution of other kinases in the assay. For example,determination of relative inhibitory activity of the kinase incomparison to recombinant SOK-1 and/or mst-3, expressed and isolated ina similar manner, assayed under similar conditions, provides selectivitydata.

Example VII

high throughput screening protocol

Test compounds Test compounds are prepared in advance from 2.5 mg/mlstock solutions in DMSO by diluting 1:10 in distilled water and then1:10 again. Ten (10) μl of the 1:100 dilution solutions (25 μg/ml in 1%DMSO) are prepared in 96 well Microlite 1 plates (Dynatech) and platesare stored at -20° C. until the evening prior to the start of the assay.

Control plates

A plate containing control solutions is included in each run of thescreen for QA purposes. Such plates are prepared at the beginning of theHTS campaign and stored at -20° C. until required. Zero inhibition (MAX.signal) wells (columns 3, 6, 8 and 10) contain 10 μl of 1% (v/v) DMSOsolution in MilliQ water. 100% inhibition (MIN signal) wells (columns1,4,9 and 11) contain 10 μl of 220 nM ZM333141/1 in 1% DMSO solution inMilliQ water. 50% inhibition (REF. signal) wells (columns 2, 5, 7 and12) contain a reference compound at a concentration known to provideapproximately 50% inhibition in 1% (v/v) DMSO solution in MilliQ water.

Assay components

(1) recombinant kinase (expressed in E. coli or eukaryotic cells asdescribed herein) or a lysate of a prokaryotic or eukaryotic cellexpressing recombinant enzyme, or the natural enzyme partially purifiedfrom a human cell line.

(2) [γ-³³ -P]-adenosine triphosphate

(3) myelin basic protein linked to the surface of PVT SPA beads(purchased from Amersham International) by an antibody-protein A orother appropriate method.

To Microlite I plates containing 10 μl of test compound, which have beenleft on the bench overnight to reach room temperature, 25 ml ofGST-Rb/ATP/ATP³³ is added, immediately followed by 20 μl of Enzyme,using two Multidrops. The plates are stacked in 13 plate stacks (with anempty plate on top of each stack to minimise evaporation from the topplate) and left at room temperature for 105 minutes. 150 μl of "StopSolution" containing beads antibody and EDTA is added using a Multidrop.The plates are sealed with Topseal-S plate sealers and left on the benchovernight, surrounded by Scotlab perspex screens. The plates are thencentrifuged (Heraeus Megafuge 3.0 R) at 2500 rpm, 1124 xg., for 5minutes (2 plates per trunnion) and counted on a Topcount (I4.34);(isotope:P³³ ; counting time: 20 seconds/well).

The data may be analysed using well-known software systems. A thresholdfor inhibition is set, e.g., 60% inhibition of scintillation signal.Compounds reaching the inhibition threshold are scored as active.

Example VIII

PCR of SEQ ID NO:1 from various human cdnas of hematopoietic origin

SEQ ID NO:1 message is determined to be present in other cells ofhematopoietic orign by PCR with cDNA's isolated from differentcell-lines of hematopoietic orign.

Target product was amplified from 2-2.5 ng of reverse transcribed mRNAsin a 20 μL reaction using Advantage™ KlenTaq polymerase (Clontech#8417-1, lot 7020348) according to the manufacturer's recommendations.Primer set sequences are sense 5'-GTCTATGATAATCACACCTGC-3' (SEQ IDNO:9), and antisense 5'-GGCACATGGAGCTCATGGGTTAAGC-3' (SEQ ID NO:10). A289 bp PCR amplicon indicative of expression of the novel kinase isgenerated derived from the 3' end of the open reading frame and 3'UTRregion of the novel cDNA (SEQ ID NO:1 base positions 1240-1528). The PCRconditions used are: 94° C. for 1 minute followed by 20 cycles of 94° C.for 50 seconds, 65° C. for 50 seconds, 72° C. for 2 minutes. 10 μL ofeach reaction is analyzed on a 1% agarose gel containing 0.5 μg/mL finalconcentration ethidium bromide in TAE buffer as in Sambrook et al.,Molecular Cloning Lab Manual, Second Edition, Cold Spring Harbor Press(1989), using 600 ng of 1 kb DNA ladder as markers (Life Technologies,cat #15615-016).

Example IX

expression and purification of gst/human signal transduction kinasefusion protein

A single, isolated, BL21 transformed clone was grown overnight in 10 mLLennox L broth (LB broth containing 50 mg/ml carbenicillin) and thenseeded into 1 liter LB broth/carbenicillin and grown at 37° C. withshaking (225 rpm) to an A₆₀₀ of 0.5-0.8. Expression of GST/kinase fusionprotein was induced by adding isopropylthio-β-galactoside to 100 mM andcontinuing the incubation for 2 additional hours. Following incubation,the cells were centrifuged 1500× g for 10 min at 4° C., resuspended in50 mL phosphate buffered saline (PBS) containing Complete™ ProteaseInhibitor Cocktail (Boehringer Mannheim GmbH), then lysed by sonicationon ice. Triton X-100 was added to the sonicate to a final concentrationof 1% to aid in the solubilization of the fusion protein. Cellulardebris was removed by centrifugation (12,000× g, 4° C.) and thesupernatant was used as the source for obtaining purified GST/kinase.

purification of gst/kinase fusion protein

GST/kinase was purified by Glutathione Sepharose 4 B beads (Pharmacia)affinity column chromatography using a 1 ml gravity fed open column. Thesuspension was allowed to pass through the column then the column waswashed three times with PBS containing protease inhibitors. Finally, theGST-kinase fusion protein was eluted from the column by the addition of1 mL elution buffer (10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0with protease inhibitors). The GST/kinase was stored in aliquots at -20°C. until needed.

Example X

dominant negative/inactive kinase mutant

A dominant negative/inactive kinase mutant, for example, can readily beproduced with the tools provided herein to further produce oneembodiment exemplified herein by changing the lysine at SEQ ID NO:3position 53 to an arginine. This mutation has been shown to produceinactive and/or dominant negative kinases for numerous otherserine/threonine kinases. See, e.g., Hanks, S. K., et al. Science,241:42 (1988). A dominant negative or inactive kinase mutant of thenovel kinase was produced by changing the lysine at position 53 to anarginine. The mutation was introduced into the novel kinase/pCR2.1construct using the Quick Change Site Directed Mutagenesis Kit(Stratagene) and the oligonucleotides5'-CAGCAAGTCGTTGCTATCCGGATCATAGACCTTGAG-3' (sense) (SEQ ID NO:13) and5'-CTCAAGGTCTATGATCCGGATAGCAACGACTTGCTG-3' (antisense) (SEQ ID NO:14).This produced the construct K53R/pCR2.1. Epitope tagged dominantnegative vector construction: A constitutive expression constructcontaining the novel kinase K53R dominant negative kinase CDS wasproduced using pcDNA3.1His (Invitrogen Corporation). This expressionconstruct is designed to express an N-terminal epitope tagged fusionprotein containing the following protein sequence:Methionine--(Histidine)₆ --Enterokinase protease cleavage site/Xpress™antibody epitope--novel kinase K53R dominant negative kinase protein.The novel kinase K53R cDNA was excised from the K53R/pCR2.1 constructusing EcoRI (5') and EcoICRI (3') and inserted into the pcDNA3.1Hisvector using EocRI (5') and EcoRV (3') restriction endonuclease sites atthe 5'end and 3'end, respectively.

Example XI

activation of transcription factors by TEN-1 kinase

Shown below is the general format of the transcription activationreporter assays used to determine if the signal transduction kinase (SEQID NO:3) activates specific transfactors. The PathDetect™ System(including reporter constructs and positive control constructs) was usedto perform these assays. Stratagene, La Jolla, Calif. All assays wereperformed in triplicate and repeated several separate times.

1. The cells were diluted to 1.5×10⁵ /ml and 2 ml (3×10⁵ cells) wereplated into each well of 6-well dishes

2. The cells were cultured overnight at 37° C., 5% CO₂, 90% relativehumitidity (these conditions were used throughout the assay

3. Cells were transfected using Stratagene's Lipotaxi reagent accordingto the protocol supplied with the reagent. The protocol is as follows(described for 3 transfections)

4. Combine 840 μl DMEM+60 μl Lipotaxi reagent in a polystyrene tube

5. Add DNA's (see table) to the DMEM/Lipotaxi solution

6. Incubate for 15-30 minutes at room temperature

7. Add 2.1 ml DMEM to the DMEM, Lipotaxi, DNA solution

8. Remove the culture media from the cells

9. Add 1 ml of the Lipid/DNA solution to each well of cells in a dropwise fashion while swirling the dish

10. Incubate the Lipid/DNA:cell cutlure for 5-7 hours at 37° C. in 5%CO₂

11. Add 1 ml DMEM/1% FBS and culture overnight

12. Replace the media with fresh DMEM/0.5% FBS and culture overnight

13. Test the cells for luciferase activity using Packard's Luc-Lite kitreagents and protocol as follows

14. Remove the media from the cells and add 300 μl of Luc-Lite reagentthat was previously diluted 1:1 with PBS containing 1 mM CaCl₂ and MgCl₂

15. Incubate at room temperature for 10 minutes

16. Dispense 200 μl of the cell lysate into white plastic 96-well platesand read the light emission on a Perkin-Elmer/Tropix 96-well Flourimeter

    __________________________________________________________________________    Table of DNA's used in a PathDetect ™ assay (TEN1 = SEQ ID NO:3)                    Positive                Expression Vector w/o Insert                                                               Carrier Vector                    Reporter Vector Control Vector, Kinase Expression Vector @ 50 ng, 250                                                     ng, 500 ng, DNA                   @ 1 μg/well @ 50 ng/well @ 50 ng, 250 ng, 500 ng, 750 ng/well 750                                                      ng/well @ 1 μg/well          __________________________________________________________________________    pX-Luc, 3 μg, 3 μl                                                               --     --               --           3 μg, 3 μl                  pX-Luc, 3 μg, 3 μl -- -- pIRES-EGFP, 150 ng, 3 μl 2.85 μg,                                                    2.85 μl                        pX-Luc, 3 μg, 3 μl -- -- pIRES-EGFP, 750 ng, 3 μl 2.25 μg,                                                    2.25 μl                        pX-Luc, 3 μg, 3 μl -- -- pIRES-EGFP, 1500 ng, 3 μl 1.5 μg,                                                    1.5 μl                         pX-Luc, 3 μg, 3 μl -- -- pIRES-EGFP, 2250 ng, 3 μl 0.75 μg,                                                   0.75 μl                        pX-Luc, 3 μg, 3 μl -- TEN1#1/pIRES-EGFP, 150 ng, 3 μl -- 2.85                                                    μg, 2.85 μl                 pX-Luc, 3 μg, 3 μl -- TEN1#1/pIRES-EGFP, 750 ng, 3 μl -- 2.25                                                    μg, 2.25 μl                 pX-Luc, 3 μg, 3 μl -- TEN1#1/pIRES-EGFP, 1500 ng, 3 μl -- 1.5                                                    μg, 1.5 μl                  pX-Luc, 3 μg, 3 μl -- TEN1#1/pIRES-EGFP, 2250 ng, 3 μl -- 0.75                                                   μg, 0.75 μl                 pX-Luc, 3 μg, 3 μl pFC-X  -- 2.85 μg, 2.85 μl                      150 ng, 6 μl                                                            __________________________________________________________________________

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described methods and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 14                                       - - <210> SEQ ID NO 1                                                        <211> LENGTH: 3201                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 1                                                         - - taacagccca cctcctagcc ccgggctacg cgccgccagc ccagtaaccc ca -            #cttttgtg     60                                                                 - - tgtcctccca ggccccgatc gaaaagcctg ggagggccgc cgaactaccc cc -            #ggagggag    120                                                                 - - gagccagtcc gaacccaagg cgccaccgcc gcagaagcgg agcgaggcag ca -            #ttcgcctc    180                                                                 - - catggcccac tcgccggtgg ctgtccaagt gcctgggatg cagaataaca ta -            #gctgatcc    240                                                                 - - agaagaactg ttcacaaaat tagagcgcat tgggaaaggc tcatttgggg aa -            #gttttcaa    300                                                                 - - aggaattgat aaccgtaccc agcaagtcgt tgctattaaa atcatagacc tt -            #gaggaagc    360                                                                 - - cgaagatgaa atagaagaca ttcagcaaga aataactgtc ttgagtcaat gt -            #gacagctc    420                                                                 - - atatgtaaca aaatactatg ggtcatattt aaaggggtct aaattatgga ta -            #ataatgga    480                                                                 - - atacctgggc ggtggttcag cactggatct tcttcgagct ggtccatttg at -            #gagttcca    540                                                                 - - gattgctacc atgctaaagg aaattttaaa aggtctggac tatctgcatt ca -            #gaaaagaa    600                                                                 - - aattcaccga gacataaaag ctgccaatgt cttgctctca gaacaaggag at -            #gttaaact    660                                                                 - - tgctgatttt ggagttgctg gtcagctgac agatacacag attaaaagaa at -            #acctttgt    720                                                                 - - gggaactcca ttttggatgg ctcctgaagt tattcaacag tcagcttatg ac -            #tcaaaagc    780                                                                 - - tgacatttgg tcattgggaa ttactgctat tgaactagcc aagggagagc ca -            #cctaactc    840                                                                 - - cgatatgcat ccaatgagag ttctgtttct tattcccaaa aacaatcctc ca -            #actcttgt    900                                                                 - - tggagacttt actaagtctt ttaaggagtt tattgatgct tgcctgaaca aa -            #gatccatc    960                                                                 - - atttcgtcct acagcaaaag aacttctgaa acacaaattc attgtaaaaa at -            #tcaaagaa   1020                                                                 - - gacttcttat ctgactgaac tgatagatcg ttttaagaga tggaaggcag aa -            #ggacacag   1080                                                                 - - tgatgatgaa tctgattccg agggctctga ttcggaatct accagcaggg aa -            #aacaatac   1140                                                                 - - tcatcctgaa tggagcttta ccaccgtacg aaagaagcct gatccaaaga aa -            #gtacagaa   1200                                                                 - - tggggcagag caagatcttg tgcaaaccyt gagttgtttg tctatgataa tc -            #acacctgc   1260                                                                 - - atttgctgaa cttaaacagc aggacgagaa taacgctagc aggaatcagg cg -            #attgaaga   1320                                                                 - - actcgagaaa agtattgctg tggctgaagc cgcctgtccc ggcatcacag at -            #aaaatggt   1380                                                                 - - gaagaaacta attgaaaaat ttcaaaagtg ttcagcagac gaatccccct aa -            #gaaactta   1440                                                                 - - ttattggctt ctgtttcata tggacccaga gagccccacc aaacctacgt ca -            #agattaac   1500                                                                 - - aatgcttaac ccatgagctc catgtgcctt ttggatcttt gcaacactga ag -            #atttggaa   1560                                                                 - - gaagctatta aactattttg tgatggcgtt tatcatttta tattttgaaa gg -            #attatttt   1620                                                                 - - gtaaggaata acttttaata ctatagtttc acctgtattc tagtaaatgt tg -            #agacaccg   1680                                                                 - - ttttgctttt aagtatccct atttcttaag ttacgaggat gaataccttt ca -            #cattttga   1740                                                                 - - tctttagttg actctacagt catgaaacat acaggtcttt caaagtcatt ct -            #caatattc   1800                                                                 - - agcttttgta aattatcaag cttcaaaaag ctttttttta aaaaaaaaaa ca -            #tgcatatt   1860                                                                 - - ctaaaaatga ctattgggtg gggaggtgta aataagtcat accttcttaa aa -            #cagaaaat   1920                                                                 - - ttaagtaaag tcttttaaat gaaacctgta aaagtattga ctcttctacc aa -            #gttggtat   1980                                                                 - - gatattccag gcagctcaat gattatcaca tttgagaccc tgtgtttgaa gc -            #atttacag   2040                                                                 - - gcaatgtaca gcaacagagg tacctcttgg tgtatagtat ttacattctc tt -            #ttaggtag   2100                                                                 - - aagaggcaat tttaccctta tttcacatgg ttagaaattt aaagcaagat ca -            #tttaccca   2160                                                                 - - aggataggtg tttggtaatg ttgaaggagt tagtctggct tcatgtttta ca -            #tcttcaac   2220                                                                 - - taaaatccca tactatctgc ttggatttgg agagccaaaa aataaagctg at -            #tgtcatgt   2280                                                                 - - gattaaatat ctgatcaaca ggtatgaata taacttaaat cagcatattt tt -            #gccatggt   2340                                                                 - - aataaattgt cctataaact atttatatat ttttgttctt cataattatc ac -            #taataagc   2400                                                                 - - atcagtttgt tgtttttaaa aggatattta agtgagcatt ttctagttca ta -            #tgaaaata   2460                                                                 - - accatagtac aggatgattt ctgtccacac aaaggttaaa ttagattgca ca -            #gttaattt   2520                                                                 - - tcacttatat ttatggtact attatgtggg tgatgccttt ttcttttaag cc -            #cagtacat   2580                                                                 - - atattatgcc tgcctaagtt ctgaactggg gctgtatttc agtagttgta ga -            #attattga   2640                                                                 - - tatttagttt tgatagctaa tgtttaattg tttggatctg cacagtttgg tt -            #tttgcaca   2700                                                                 - - aaagtcattt aaaaaaatct gagtaattgt caaatattaa aagaaagata tt -            #cttcctgt   2760                                                                 - - aaggaataca gtttttagtc aaagtggcca ttacatcctc tttttaattt ac -            #ataataca   2820                                                                 - - gatacttgag aaagttgttg tggtgttgta tgccaagaaa attcttttta tt -            #ggtgccta   2880                                                                 - - tattgtaaca attattttta atgcattgta ttttgaagta acggttcagt ta -            #aatttttc   2940                                                                 - - acctgctgtg taactgaaac acaattacag tttataatca tctgtagaag tc -            #tggagata   3000                                                                 - - attttgcaac tcatgttatg ggttaaatga atatttttgt aaaagtaaaa gc -            #aacaaatt   3060                                                                 - - tataaattga ttatttgaaa ctttacaaca caattgcatc ccaaatacaa at -            #tgtattgc   3120                                                                 - - ttattcatta tagctattcg tcctgtaatc tgtttctagg tgaagcatac tc -            #cagtgttt   3180                                                                 - - taggggtttt gaaaataaat a           - #                  - #                    3201                                                                     - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 1251                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 2                                                         - - atggcccact cgccggtggc tgtccaagtg cctgggatgc agaataacat ag -             #ctgatcca     60                                                                 - - gaagaactgt tcacaaaatt agagcgcatt gggaaaggct catttgggga ag -            #ttttcaaa    120                                                                 - - ggaattgata accgtaccca gcaagtcgtt gctattaaaa tcatagacct tg -            #aggaagcc    180                                                                 - - gaagatgaaa tagaagacat tcagcaagaa ataactgtct tgagtcaatg tg -            #acagctca    240                                                                 - - tatgtaacaa aatactatgg gtcatattta aaggggtcta aattatggat aa -            #taatggaa    300                                                                 - - tacctgggcg gtggttcagc actggatctt cttcgagctg gtccatttga tg -            #agttccag    360                                                                 - - attgctacca tgctaaagga aattttaaaa ggtctggact atctgcattc ag -            #aaaagaaa    420                                                                 - - attcaccgag acataaaagc tgccaatgtc ttgctctcag aacaaggaga tg -            #ttaaactt    480                                                                 - - gctgattttg gagttgctgg tcagctgaca gatacacaga ttaaaagaaa ta -            #cctttgtg    540                                                                 - - ggaactccat tttggatggc tcctgaagtt attcaacagt cagcttatga ct -            #caaaagct    600                                                                 - - gacatttggt cattgggaat tactgctatt gaactagcca agggagagcc ac -            #ctaactcc    660                                                                 - - gatatgcatc caatgagagt tctgtttctt attcccaaaa acaatcctcc aa -            #ctcttgtt    720                                                                 - - ggagacttta ctaagtcttt taaggagttt attgatgctt gcctgaacaa ag -            #atccatca    780                                                                 - - tttcgtccta cagcaaaaga acttctgaaa cacaaattca ttgtaaaaaa tt -            #caaagaag    840                                                                 - - acttcttatc tgactgaact gatagatcgt tttaagagat ggaaggcaga ag -            #gacacagt    900                                                                 - - gatgatgaat ctgattccga gggctctgat tcggaatcta ccagcaggga aa -            #acaatact    960                                                                 - - catcctgaat ggagctttac caccgtacga aagaagcctg atccaaagaa ag -            #tacagaat   1020                                                                 - - ggggcagagc aagatcttgt gcaaaccctg agttgtttgt ctatgataat ca -            #cacctgca   1080                                                                 - - tttgctgaac ttaaacagca ggacgagaat aacgctagca ggaatcaggc ga -            #ttgaagaa   1140                                                                 - - ctcgagaaaa gtattgctgt ggctgaagcc gcctgtcccg gcatcacaga ta -            #aaatggtg   1200                                                                 - - aagaaactaa ttgaaaaatt tcaaaagtgt tcagcagacg aatcccccta a - #               1251                                                                        - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 416                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 3                                                         - - Met Ala His Ser Pro Val Ala Val Gln Val Pr - #o Gly Met Gln Asn Asn       1               5  - #                10  - #                15               - - Ile Ala Asp Pro Glu Glu Leu Phe Thr Lys Le - #u Glu Arg Ile Gly Lys                  20      - #            25      - #            30                   - - Gly Ser Phe Gly Glu Val Phe Lys Gly Ile As - #p Asn Arg Thr Gln Gln              35          - #        40          - #        45                       - - Val Val Ala Ile Lys Ile Ile Asp Leu Glu Gl - #u Ala Glu Asp Glu Ile          50              - #    55              - #    60                           - - Glu Asp Ile Gln Gln Glu Ile Thr Val Leu Se - #r Gln Cys Asp Ser Ser      65                  - #70                  - #75                  - #80        - - Tyr Val Thr Lys Tyr Tyr Gly Ser Tyr Leu Ly - #s Gly Ser Lys Leu Trp                      85  - #                90  - #                95               - - Ile Ile Met Glu Tyr Leu Gly Gly Gly Ser Al - #a Leu Asp Leu Leu Arg                  100      - #           105      - #           110                  - - Ala Gly Pro Phe Asp Glu Phe Gln Ile Ala Th - #r Met Leu Lys Glu Ile              115          - #       120          - #       125                      - - Leu Lys Gly Leu Asp Tyr Leu His Ser Glu Ly - #s Lys Ile His Arg Asp          130              - #   135              - #   140                          - - Ile Lys Ala Ala Asn Val Leu Leu Ser Glu Gl - #n Gly Asp Val Lys Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Asp Phe Gly Val Ala Gly Gln Leu Thr As - #p Thr Gln Ile Lys        Arg                                                                                             165  - #               170  - #               175             - - Asn Thr Phe Val Gly Thr Pro Phe Trp Met Al - #a Pro Glu Val Ile Gln                  180      - #           185      - #           190                  - - Gln Ser Ala Tyr Asp Ser Lys Ala Asp Ile Tr - #p Ser Leu Gly Ile Thr              195          - #       200          - #       205                      - - Ala Ile Glu Leu Ala Lys Gly Glu Pro Pro As - #n Ser Asp Met His Pro          210              - #   215              - #   220                          - - Met Arg Val Leu Phe Leu Ile Pro Lys Asn As - #n Pro Pro Thr Leu Val      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Gly Asp Phe Thr Lys Ser Phe Lys Glu Phe Il - #e Asp Ala Cys Leu        Asn                                                                                             245  - #               250  - #               255             - - Lys Asp Pro Ser Phe Arg Pro Thr Ala Lys Gl - #u Leu Leu Lys His Lys                  260      - #           265      - #           270                  - - Phe Ile Val Lys Asn Ser Lys Lys Thr Ser Ty - #r Leu Thr Glu Leu Ile              275          - #       280          - #       285                      - - Asp Arg Phe Lys Arg Trp Lys Ala Glu Gly Hi - #s Ser Asp Asp Glu Ser          290              - #   295              - #   300                          - - Asp Ser Glu Gly Ser Asp Ser Glu Ser Thr Se - #r Arg Glu Asn Asn Thr      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - His Pro Glu Trp Ser Phe Thr Thr Val Arg Ly - #s Lys Pro Asp Pro        Lys                                                                                             325  - #               330  - #               335             - - Lys Val Gln Asn Gly Ala Glu Gln Asp Leu Va - #l Gln Thr Leu Ser Cys                  340      - #           345      - #           350                  - - Leu Ser Met Ile Ile Thr Pro Ala Phe Ala Gl - #u Leu Lys Gln Gln Asp              355          - #       360          - #       365                      - - Glu Asn Asn Ala Ser Arg Asn Gln Ala Ile Gl - #u Glu Leu Glu Lys Ser          370              - #   375              - #   380                          - - Ile Ala Val Ala Glu Ala Ala Cys Pro Gly Il - #e Thr Asp Lys Met Val      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Lys Lys Leu Ile Glu Lys Phe Gln Lys Cys Se - #r Ala Asp Glu Ser        Pro                                                                                             405  - #               410  - #               415             - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 426                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 4                                                         - - Met Ala His Leu Arg Gly Phe Ala Asn Gln Hi - #s Ser Arg Val Asp Pro       1               5  - #                10  - #                15               - - Glu Glu Leu Phe Thr Lys Leu Asp Arg Ile Gl - #y Lys Gly Ser Phe Gly                  20      - #            25      - #            30                   - - Glu Val Tyr Lys Gly Ile Asp Asn His Thr Ly - #s Glu Val Val Ala Ile              35          - #        40          - #        45                       - - Lys Ile Ile Asp Leu Glu Glu Ala Glu Asp Gl - #u Ile Glu Asp Ile Gln          50              - #    55              - #    60                           - - Gln Glu Ile Thr Val Leu Ser Gln Cys Asp Se - #r Pro Tyr Ile Thr Arg      65                  - #70                  - #75                  - #80        - - Tyr Phe Gly Ser Tyr Leu Lys Ser Thr Lys Le - #u Trp Ile Ile Met Glu                      85  - #                90  - #                95               - - Tyr Leu Gly Gly Gly Ser Ala Leu Asp Leu Le - #u Lys Pro Gly Pro Leu                  100      - #           105      - #           110                  - - Glu Glu Thr Tyr Ile Ala Thr Ile Leu Arg Gl - #u Ile Leu Lys Gly Leu              115          - #       120          - #       125                      - - Asp Tyr Leu His Ser Glu Arg Lys Ile His Ar - #g Asp Ile Lys Ala Ala          130              - #   135              - #   140                          - - Asn Val Leu Leu Ser Glu Gln Gly Asp Val Ly - #s Leu Ala Asp Phe Gly      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Val Ala Gly Gln Leu Thr Asp Thr Gln Ile Ly - #s Arg Asn Thr Phe        Val                                                                                             165  - #               170  - #               175             - - Gly Thr Pro Phe Trp Met Ala Pro Glu Val Il - #e Lys Gln Ser Ala Tyr                  180      - #           185      - #           190                  - - Asp Phe Lys Ala Asp Ile Trp Ser Leu Gly Il - #e Thr Ala Ile Glu Leu              195          - #       200          - #       205                      - - Ala Lys Gly Glu Pro Pro Asn Ser Asp Leu Hi - #s Pro Met Arg Val Leu          210              - #   215              - #   220                          - - Phe Leu Ile Pro Lys Asn Ser Pro Pro Thr Le - #u Glu Gly Gln His Ser      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Lys Pro Phe Lys Glu Phe Val Glu Ala Cys Le - #u Asn Lys Asp Pro        Arg                                                                                             245  - #               250  - #               255             - - Phe Arg Pro Thr Ala Lys Glu Leu Leu Lys Hi - #s Lys Phe Ile Thr Arg                  260      - #           265      - #           270                  - - Tyr Thr Lys Lys Thr Ser Phe Leu Thr Glu Le - #u Ile Asp Arg Tyr Lys              275          - #       280          - #       285                      - - Arg Trp Lys Ser Glu Gly His Gly Glu Glu Se - #r Ser Ser Glu Asp Ser          290              - #   295              - #   300                          - - Asp Ile Asp Gly Glu Ala Glu Asp Gly Glu Gl - #n Gly Pro Ile Trp Thr      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Phe Pro Pro Thr Ile Arg Pro Ser Pro His Se - #r Lys Leu His Lys        Gly                                                                                             325  - #               330  - #               335             - - Thr Ala Leu His Ser Ser Gln Lys Pro Ala As - #p Ala Val Lys Arg Gln                  340      - #           345      - #           350                  - - Pro Arg Ser Gln Cys Leu Ser Thr Leu Val Ar - #g Pro Val Phe Gly Glu              355          - #       360          - #       365                      - - Leu Lys Glu Lys His Lys Gln Ser Gly Gly Se - #r Val Gly Ala Leu Glu          370              - #   375              - #   380                          - - Glu Leu Glu Asn Ala Phe Ser Leu Ala Glu Gl - #u Ser Cys Pro Gly Ile      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Ser Asp Lys Leu Met Val His Leu Val Glu Ar - #g Val Gln Arg Phe        Ser                                                                                             405  - #               410  - #               415             - - His Asn Arg Asn His Leu Thr Ser Thr Arg                                              420      - #           425                                         - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 431                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 5                                                         - - Met Ala His Ser Pro Val Gln Ser Gly Leu Pr - #o Gly Met Gln Asn Leu       1               5  - #                10  - #                15               - - Lys Ala Asp Pro Glu Glu Leu Phe Thr Lys Le - #u Glu Lys Ile Gly Lys                  20      - #            25      - #            30                   - - Gly Ser Phe Gly Glu Val Phe Lys Gly Ile As - #p Asn Arg Thr Gln Lys              35          - #        40          - #        45                       - - Val Val Ala Ile Lys Ile Ile Asp Leu Glu Gl - #u Ala Glu Asp Glu Ile          50              - #    55              - #    60                           - - Glu Asp Ile Gln Gln Glu Ile Thr Val Leu Se - #r Gln Cys Asp Ser Pro      65                  - #70                  - #75                  - #80        - - Tyr Val Thr Lys Tyr Tyr Gly Ser Tyr Leu Ly - #s Asp Thr Lys Leu Trp                      85  - #                90  - #                95               - - Ile Ile Met Glu Tyr Leu Gly Gly Gly Ser Al - #a Leu Asp Leu Leu Glu                  100      - #           105      - #           110                  - - Pro Gly Pro Leu Asp Glu Thr Gln Ile Ala Th - #r Ile Leu Arg Glu Ile              115          - #       120          - #       125                      - - Leu Lys Gly Leu Asp Tyr Leu His Ser Glu Ly - #s Lys Ile His Arg Asp          130              - #   135              - #   140                          - - Ile Lys Ala Ala Asn Val Leu Leu Ser Glu Hi - #s Gly Glu Val Lys Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Asp Phe Gly Val Ala Gly Gln Leu Thr As - #p Thr Gln Ile Lys        Arg                                                                                             165  - #               170  - #               175             - - Asn Thr Phe Val Gly Thr Pro Phe Trp Met Al - #a Pro Glu Val Ile Lys                  180      - #           185      - #           190                  - - Gln Ser Ala Tyr Asp Ser Lys Ala Asp Ile Tr - #p Ser Leu Gly Ile Thr              195          - #       200          - #       205                      - - Ala Ile Glu Leu Ala Arg Gly Glu Pro Pro Hi - #s Ser Glu Leu His Pro          210              - #   215              - #   220                          - - Met Lys Val Leu Phe Leu Ile Pro Lys Asn As - #n Pro Pro Thr Leu Glu      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Gly Asn Tyr Ser Lys Pro Leu Lys Glu Phe Va - #l Glu Ala Cys Leu        Asn                                                                                             245  - #               250  - #               255             - - Lys Glu Pro Ser Phe Arg Pro Thr Ala Lys Gl - #u Leu Leu Lys His Lys                  260      - #           265      - #           270                  - - Phe Ile Leu Arg Asn Ala Lys Lys Thr Ser Ty - #r Leu Thr Glu Leu Ile              275          - #       280          - #       285                      - - Asp Arg Tyr Lys Arg Trp Lys Ala Glu Gln Se - #r His Asp Asp Ser Ser          290              - #   295              - #   300                          - - Ser Glu Asp Ser Asp Ala Glu Thr Asp Gly Gl - #n Ala Ser Gly Gly Ser      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Asp Ser Gly Asp Trp Ile Phe Thr Ile Arg Gl - #u Lys Asp Pro Lys        Asn                                                                                             325  - #               330  - #               335             - - Leu Glu Asn Gly Ala Leu Gln Pro Ser Asp Le - #u Asp Arg Asn Lys Met                  340      - #           345      - #           350                  - - Lys Asp Ile Pro Lys Arg Pro Phe Ser Gln Cy - #s Leu Ser Thr Ile Ile              355          - #       360          - #       365                      - - Ser Pro Leu Phe Ala Glu Leu Lys Glu Lys Se - #r Gln Ala Cys Gly Gly          370              - #   375              - #   380                          - - Asn Leu Gly Ser Ile Glu Glu Leu Arg Gly Al - #a Ile Tyr Leu Ala Glu      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Glu Val Cys Pro Gly Ile Ser Asp Thr Met Va - #l Ala Gln Leu Val        Gln                                                                                             405  - #               410  - #               415             - - Arg Leu Gln Arg Tyr Ser Leu Ser Gly Gly Gl - #y Thr Ser Ser His                      420      - #           425      - #           430                  - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR primer                                            - - <400> SEQUENCE: 6                                                         - - gcctccatgg cccactcgcc ggtg          - #                  - #                    24                                                                      - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR primer                                            - - <400> SEQUENCE: 7                                                         - - ggcacatgga gctcatgggt taagc          - #                  - #                   25                                                                      - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 1353                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 8                                                         - - gcctccatgg cccactcgcc ggtggctgtc caagtgcctg ggatgcagaa ta -             #acatagct     60                                                                 - - gatccagaag aactgttcac aaaattagag cgcattggga aaggctcatt tg -            #gggaagtt    120                                                                 - - ttcaaaggaa ttgataaccg tacccagcaa gtcgttgcta ttaaaatcat ag -            #accttgag    180                                                                 - - gaagccgaag atgaaataga agacattcag caagaaataa ctgtcttgag tc -            #aatgtgac    240                                                                 - - agctcatatg taacaaaata ctatgggtca tatttaaagg ggtctaaatt at -            #ggataata    300                                                                 - - atggaatacc tgggcggtgg ttcagcactg gatcttcttc gagctggtcc at -            #ttgatgag    360                                                                 - - ttccagattg ctaccatgct aaaggaaatt ttaaaaggtc tggactatct gc -            #attcagaa    420                                                                 - - aagaaaattc accgagacat aaaagctgcc aatgtcttgc tctcagaaca ag -            #gagatgtt    480                                                                 - - aaacttgctg attttggagt tgctggtcag ctgacagata cacagattaa aa -            #gaaatacc    540                                                                 - - tttgtgggaa ctccattttg gatggctcct gaagttattc aacagtcagc tt -            #atgactca    600                                                                 - - aaagctgaca tttggtcatt gggaattact gctattgaac tagccaaggg ag -            #agccacct    660                                                                 - - aactccgata tgcatccaat gagagttctg tttcttattc ccaaaaacaa tc -            #ctccaact    720                                                                 - - cttgttggag actttactaa gtcttttaag gagtttattg atgcttgcct ga -            #acaaagat    780                                                                 - - ccatcatttc gtcctacagc aaaagaactt ctgaaacaca aattcattgt aa -            #aaaattca    840                                                                 - - aagaagactt cttatctgac tgaactgata gatcgtttta agagatggaa gg -            #cagaagga    900                                                                 - - cacagtgatg atgaatctga ttccgagggc tctgattcgg aatctaccag ca -            #gggaaaac    960                                                                 - - aatactcatc ctgaatggag ctttaccacc gtacgaaaga agcctgatcc aa -            #agaaagta   1020                                                                 - - cagaatgggg cagagcaaga tcttgtgcaa accctgagtt gtttgtctat ga -            #taatcaca   1080                                                                 - - cctgcatttg ctgaacttaa acagcaggac gagaataacg ctagcaggaa tc -            #aggcgatt   1140                                                                 - - gaagaactcg agaaaagtat tgctgtggct gaagccgcct gtcccggcat ca -            #cagataaa   1200                                                                 - - atggtgaaga aactaattga aaaatttcaa aagtgttcag cagacgaatc cc -            #cctaagaa   1260                                                                 - - acttattatt ggcttctgtt tcatatggac ccagagagcc ccaccaaacc ta -            #cgtcaaga   1320                                                                 - - ttaacaatgc ttaacccatg agctccatgt gcc       - #                  -      #       1353                                                                     - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR primer                                            - - <400> SEQUENCE: 9                                                         - - gtctatgata atcacacctg c           - #                  - #                      - #21                                                                  - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR primer                                            - - <400> SEQUENCE: 10                                                        - - ggcacatgga gctcatgggt taagc          - #                  - #                   25                                                                      - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 416                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Mus musculus                                                   - - <400> SEQUENCE: 11                                                        - - Met Ala His Ser Pro Val Ala Val Gln Val Pr - #o Gly Met Gln Asn Asn       1               5  - #                10  - #                15               - - Ile Ala Asp Pro Glu Glu Leu Phe Thr Lys Le - #u Glu Arg Ile Gly Lys                  20      - #            25      - #            30                   - - Gly Ser Phe Gly Glu Val Phe Lys Gly Ile As - #p Asn Arg Thr Gln Gln              35          - #        40          - #        45                       - - Val Val Ala Ile Lys Ile Ile Asp Leu Glu Gl - #u Ala Glu Asp Glu Ile          50              - #    55              - #    60                           - - Glu Asp Ile Gln Gln Glu Ile Thr Val Leu Se - #r Gln Cys Asp Ser Ser      65                  - #70                  - #75                  - #80        - - Tyr Val Thr Lys Tyr Tyr Gly Ser Tyr Leu Ly - #s Gly Ser Lys Leu Trp                      85  - #                90  - #                95               - - Ile Ile Met Glu Tyr Leu Gly Gly Gly Ser Al - #a Leu Asp Leu Leu Arg                  100      - #           105      - #           110                  - - Ala Gly Pro Phe Asp Glu Phe Gln Ile Ala Th - #r Met Leu Lys Glu Ile              115          - #       120          - #       125                      - - Leu Lys Gly Leu Asp Tyr Leu His Ser Glu Ly - #s Lys Ile His Arg Asp          130              - #   135              - #   140                          - - Ile Lys Ala Ala Asn Val Leu Leu Ser Glu Gl - #n Gly Asp Val Lys Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Asp Phe Gly Val Ala Gly Gln Leu Thr As - #p Thr Gln Ile Lys        Arg                                                                                             165  - #               170  - #               175             - - Asn Thr Phe Val Gly Thr Pro Phe Trp Met Al - #a Pro Glu Val Ile Gln                  180      - #           185      - #           190                  - - Gln Ser Ala Tyr Asp Ser Lys Ala Asp Ile Tr - #p Ser Leu Gly Ile Thr              195          - #       200          - #       205                      - - Ala Ile Glu Leu Ala Lys Gly Glu Pro Pro As - #n Ser Asp Met His Pro          210              - #   215              - #   220                          - - Met Arg Val Leu Phe Leu Ile Pro Lys Asn As - #n Pro Pro Thr Leu Ile      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Gly Asp Phe Thr Lys Ser Phe Lys Glu Phe Il - #e Asp Ala Cys Leu        Asn                                                                                             245  - #               250  - #               255             - - Lys Asp Pro Ser Phe Arg Pro Thr Ala Lys Gl - #u Leu Leu Lys His Lys                  260      - #           265      - #           270                  - - Phe Ile Val Lys Asn Ser Lys Lys Thr Ser Ty - #r Leu Thr Glu Leu Ile              275          - #       280          - #       285                      - - Asp Arg Phe Lys Arg Trp Lys Ala Glu Gly Hi - #s Ser Asp Glu Glu Ser          290              - #   295              - #   300                          - - Asp Ser Glu Gly Ser Asp Ser Glu Ser Ser Se - #r Arg Glu Ser Asn Pro      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - His Pro Glu Trp Ser Phe Thr Thr Val Arg Ly - #s Lys Pro Asp Pro        Lys                                                                                             325  - #               330  - #               335             - - Lys Leu Gln Asn Gly Glu Glu Gln Asp Leu Va - #l Gln Thr Leu Ser Cys                  340      - #           345      - #           350                  - - Leu Ser Met Ile Ile Thr Pro Ala Phe Ala Gl - #u Leu Lys Gln Gln Asp              355          - #       360          - #       365                      - - Glu Asn Asn Ala Ser Arg Asn Gln Ala Ile Gl - #u Glu Leu Glu Lys Ser          370              - #   375              - #   380                          - - Ile Ala Val Ala Glu Thr Ala Cys Pro Gly Il - #e Thr Asp Lys Met Val      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Lys Lys Leu Ile Glu Lys Phe Gln Lys Cys Se - #r Ala Asp Glu Ser        Pro                                                                                             405  - #               410  - #               415             - -  - - <210> SEQ ID NO 12                                                  <211> LENGTH: 2028                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Mus musculus                                                   - - <400> SEQUENCE: 12                                                        - - aggcaccgcc acaggtcaag ccctgcattc aggaaagaga gcaacactgc ag -             #ttagccaa     60                                                                 - - aagccaggca ggcgagcggc agagaggcct cgatcgagaa gcctggtaga gc -            #tgcagaga    120                                                                 - - tacctccgta ggaggagcca gtctctgccg gaggcgccac cgccaccacc gc -            #agaagcag    180                                                                 - - cgcgaagtag cagtcgccac catggcccac tcaccggtgg ctgttcaagt gc -            #ctgggatg    240                                                                 - - cagaataata tagcagatcc agaagaactg ttcacaaaat tagagcgcat tg -            #gaaaaggc    300                                                                 - - tcctttggag aagttttcaa aggaattgat aaccgtactc agcaagtggt tg -            #caattaaa    360                                                                 - - atcattgacc ttgaggaagc tgaggatgaa atagaagaca tccaacaaga aa -            #taactgtt    420                                                                 - - ttgagtcagt gcgacagctc atatgtaaca aaatactatg ggtcctattt aa -            #agggttca    480                                                                 - - aaactatgga taataatgga atacctaggt ggaggttcag cattggatct tc -            #tgcgtgct    540                                                                 - - ggtccatttg atgagttcca gattgccacc atgctcaagg agattttgaa ag -            #gtctggac    600                                                                 - - tatctacatt ctgaaaagaa aatccaccga gacattaaag ctgccaacgt ct -            #tgctttca    660                                                                 - - gaacaaggtg atgttaaact ggctgacttt ggagttgctg gccagctgac ag -            #atacacaa    720                                                                 - - atcaaaagaa acaccttcgt agggactccg ttttggatgg ctcctgaagt ta -            #ttcaacag    780                                                                 - - tcagcttatg actctaaagc tgacatatgg tctttgggaa ttactgctat tg -            #aacttgcc    840                                                                 - - aagggagagc ctccgaattc tgacatgcat ccaatgagag ttctgtttct ta -            #ttccaaaa    900                                                                 - - aacaaccctc caactcttat tggagacttt actaagtctt tcaaggagtt ta -            #ttgatgct    960                                                                 - - tgcctgaata aagacccgtc atttcgtcct acagctaaag aacttttgaa gc -            #ataagttc   1020                                                                 - - atcgtaaaaa attcaaagaa gacttcttat ctgactgaat tgatcgatcg at -            #ttaagaga   1080                                                                 - - tggaaggcag aaggccacag tgatgaggaa tctgattccg agggctctga ct -            #cggaatcc   1140                                                                 - - agcagcaggg aaagtaaccc tcaccctgaa tggagtttca ccactgtgcg ta -            #agaagcct   1200                                                                 - - gatccaaaga aactgcagaa tggggaagag caagatcttg tgcaaacctt ga -            #gctgtttg   1260                                                                 - - tctatgataa tcacacctgc atttgccgaa cttaaacagc aggacgagaa ta -            #atgcgagt   1320                                                                 - - cgaaaccagg caattgaaga acttgagaaa agtattgctg tggctgaaac cg -            #cctgtcct   1380                                                                 - - ggcatcacag ataagatggt gaagaaacta atcgaaaaat ttcaaaagtg tt -            #ctgcggat   1440                                                                 - - gaatcccctt aagaaatctg ttgtcattac ttttggcttc tgtttcatgt gg -            #accaggag   1500                                                                 - - aaacccacca aagctatgtc aaccttataa atgcttaact catgagctcc at -            #gtgccttt   1560                                                                 - - tggatctttg ccacattgaa gatttagagg aagctattaa actattttgt ga -            #tggtgatt   1620                                                                 - - atcattttgt attttaaaga gattattttg taaggaataa ttttaatact at -            #agttttgc   1680                                                                 - - cggtattgta gtaaatgctg agatacaggt tttttgtttt ttgtttttta at -            #tttaggta   1740                                                                 - - ccattatttc ttatgttcat ggaatgaata ctgtttggtt tggaatcttt ag -            #ttaactgt   1800                                                                 - - atactcataa acatacaggt ctttcaaagt catcctaact attaaatgtt tg -            #taaatcat   1860                                                                 - - caagcttcaa aaagcattct ttttccccca cacaagtata ttctaaaaat ga -            #ctatttgt   1920                                                                 - - aatgaggtgg aagtaagtaa taccttctta aaacaagtgt ttttaagaag ct -            #cccggaaa   1980                                                                 - - aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa  - #                  2028                                                                        - -  - - <210> SEQ ID NO 13                                                  <211> LENGTH: 36                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR primer                                            - - <400> SEQUENCE: 13                                                        - - cagcaagtcg ttgctatccg gatcatagac cttgag      - #                  -     #       36                                                                      - -  - - <210> SEQ ID NO 14                                                  <211> LENGTH: 36                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR primer                                            - - <400> SEQUENCE: 14                                                        - - ctcaaggtct atgatccgga tagcaacgac ttgctg      - #                  -     #       36                                                                    __________________________________________________________________________

What is claimed is:
 1. A purified polynucleotide comprising a nucleicacid sequence which encodes a polypeptide comprising an amino acidsequence having at least about 90% homology to a member selected fromthe group consisting of: (SEQ ID NO:3, SEQ ID NO:3 positions 24-274, andSEQ ID NO:3 positions 275-416).
 2. A polynucleotide according to claim 1comprising a nucleic acid sequence which encodes a polypeptidecomprising an amino acid sequence wherein one or more positionscorresponding to SEQ ID NO:3, selected from the group consisting of(position 31 (glycine), 33 (glycine), 36 (glycine), 38 (valine), 51(alanine), 53 (lysine), 144 (aspartic acid), 149 (asparagine), 162(aspartic acid), 163 (phenylalanine), 164 (glycine), 182 (threonine),189 (glutamic acid), and 201 (aspartic acid)), are substituted ordeleted.
 3. A polynucleotide according to claim 2 comprising a nucleicacid sequence which encodes a polypeptide comprising the amino acidsequence as depicted in SEQ ID NO:3 wherein position 53 of SEQ ID NO:3is arginine.
 4. A polynucleotide according to claim 1 comprising anucleic acid sequence which encodes a polypeptide comprising an aminoacid sequence selected from the group consisting essentially of: (SEQ IDNO:3, SEQ ID NO:3 positions 24-274, and SEQ ID NO:3 positions 275-416).5. A polynucleotide of claim 1 wherein the polynucleotide sequencecomprises the sequence as depicted in SEQ ID NO:2.
 6. An antisensemolecule comprising an oligomer in the range from about 12 to about 25nucleotides in length which: (a) is complementary to a region withinpositions 157-232 or positions 1405-1480 of SEQ ID NO:1; or, (b)comprises a sequence which is complementary to a sequence selected fromthe group consisting of: (SEQ ID NO:1 positions 157-168, 158-169,159-170, 160-171, . . . et seq (all inclusive) . . . 219-230, 220-231,and 221-232) (note: 65 species); or comprises a sequence which iscomplementary to a sequence selected from the group consisting of: (SEQID NO:1 positions 1405-1416, 1406-1417, 1407-1418, 1408-1419, . . . etseq (all inclusive) . . . 1467-1478, 1468-1479, and 1469-1480) (note: 65species).
 7. An expression vector comprising the polynucleotide ofclaim
 1. 8. A host cell transformed with the expression vector of claim7.